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human disease
by
Lev Prasov
Doctoral Committee:
Associate Professor Thomas M. Glaser, Chair
Professor Sally A. Camper
Professor Pamela A. Raymond
Associate Professor Donna M. Martin
Associate Professor David L. Turner
Lev Prasov
2012
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ACKNOWLEDGEMENTS
Many people have contributed to the work presented in this thesis. First
and foremost, I would like to thank my advisor, Tom Glaser, for his guidance and
support throughout this process. He deserves much of the credit for my success
as a graduate student, and has truly taught me to be a good and rigorous
scientist. I would like to thank my thesis committee (David Turner, Donna Martin,
Sally Camper, and Pamela Raymond) for fruitful discussions and constructive
feedback. I would like to thank all of the Glaser lab members past and present
(Chris Chou, Dellaney Rudolph, Mindy Nagy, Nate Vale, and Sherry Taylor) for
creating a vibrant and productive work environment, including the entertaining
discussions both about science and about nothing. I would also like to
acknowledge the efforts and support of all the people that have provided me with
reagents, shared ideas, assisted with experiments, and helped with manuscripts.
Foremost, I would like to thank Chris Chou, Joe Brzezinski, Dave Turner, and
Nadean Brown, whose advice and support have been truly invaluable. Id like to
thank the staff at the MSTP and HG department, especially Ron Koenig, Ellen
Elkin, and Karen Grahl, for their administrative support and advice. Id like to
thank my friends, who have helped me stay sane throughout the PhD process.
Finally, Id like to thank my parents and grandparents for their love and support,
and my brother, Zahar, for being my first and best teacher.
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PREFACE
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Masud T, Khaliq S, Mehdi SQ, Abid, AY, Oliver ER, Silva E, Brodsky MC,
Borchert M, Kelberman D, Snowden JC, Dattani M, Glaser T. Mutations in Atoh7
cause recessive persistent hyperplasia of the primary vitreous.
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TABLE OF CONTENTS
DEDICATION ....................................................................................................... ii
ACKNOWLEDGEMENTS ................................................................................... iii
PREFACE ........................................................................................................... iv
LIST OF FIGURES ............................................................................................... x
LIST OF TABLES ............................................................................................. xiv
ABSTRACT ........................................................................................................ xv
CHAPTER I: VERTEBRATE RETINAL DEVELOPMENT ................................. 1
Structure and composition of the retina ............................................................. 1
Control of retinal development........................................................................... 4
Cell divisions and the retinal cell cycle .............................................................. 6
Cell death in the retina ...................................................................................... 9
Intrinsic and extrinsic signals in retinal development ....................................... 10
Math5 (Atoh7) and control of RGC fate ........................................................... 13
Retinal vascular development and the role of RGCs ....................................... 15
Diseases of the optic nerve and retinal vasculature ........................................ 17
Novel insights into RGC development and the role of Math5 (ATOH7) ........... 20
CHAPTER II: A CRITICAL ANALYSIS OF MATH5 (ATOH7) mRNA
SPLICING IN THE DEVELOPING MOUSE RETINA ......................................... 29
Abstract ........................................................................................................... 29
Introduction...................................................................................................... 30
Materials and Methods .................................................................................... 32
Results ............................................................................................................ 40
Discussion ....................................................................................................... 50
Acknowledgements ......................................................................................... 55
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ix
LIST OF FIGURES
CHAPTER I:
Figure I-1. Structure of the eye and retina. ..................................................... 23
Figure I-2. Molecular mechanisms of retinal development ............................. 24
Figure I-3. Math5 (Atoh7) knockout mice have no optic nerves, and severe
deficiencies in RGCs ....................................................................................... 26
Figure I-4. Development of the retinal vasculature ......................................... 27
CHAPTER II:
Figure II-1. Anatomy of the Math5 transcription unit ....................................... 56
Figure II-2. Math5 messenger RNAs .............................................................. 58
Figure II-3. Math5 embryonic eye RT PCRs with increasing amounts of
betaine. ........................................................................................................... 60
Figure II-4. RT PCRs of Math5 RNA transcribed in vitro ................................ 61
Figure II-5. Triplex competitive RT PCR assay to evaluate trace levels of
Math5 splicing in the embryonic retina ........................................................... 63
Figure II-6. Ribonuclease protection assays................................................... 65
Figure II-7. Model explaining the observed results ......................................... 67
Figure II-S1. Math5 ESTs in the public domain .............................................. 68
Figure II-S2. Evaluation of Math5 antibodies. ................................................. 70
Figure II-S3. Math5 splicing in the cerebellum................................................ 72
Figure II-S4. Splicing patterns in the mouse Atonal related bHLH genes ....... 74
Figure II-S5. Secondary structure for Math5 mRNA ....................................... 75
CHAPTER III:
Figure III-1. Math5 is expressed by early retinal progenitors during
or shortly following their terminal cell cycle ................................................... 122
Figure III-2. Construction and expression of the Math5>Cre transgene ....... 124
Figure III-3. Math5+ progenitors contribute differentially to all retinal
cell types ....................................................................................................... 126
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CHAPTER IV:
Figure IV-1. Timing of cell cycle progression in the mouse retinal
neuroepithelium at E13.5 and E15.5 ............................................................. 165
Figure IV-2. Coexpression and onset analysis of amacrine and horizontal
markers Ptf1a and AP2. .............................................................................. 166
Figure IV-3. The onset of Brn3b and Isl1 expression within individual
cells is progressively delayed during retinal development. ............................ 168
Figure IV-4. Co-expression of Brn3b and Isl1 during or shortly after the
terminal cell cycle .......................................................................................... 170
Figure IV-5. Paired ganglion cells can be generated from retinal
progenitors by symmetric terminal division.................................................... 168
CHAPTER V:
Figure V-1. Crx and Math5 (gal) are expressed in overlapping subsets of
cells shortly after cell cycle exit ..................................................................... 213
Figure V-2. Characterization of the Crx>Math5-IRES-Cre and Crx>Cre
conventional and BAC transgenic mice ......................................................... 214
Figure V-3. Widespread Crx>Math5 expression has little effect on cell fate
decisions in the retina.................................................................................... 216
Figure V-4. Widespread Crx>Math5 expression does not alter RGC
abundance or retinal histology....................................................................... 217
Figure V-5. Widespread Crx>Math5 expression does not extend the profile
of RGC births, but decreases the numbers of early-born photoreceptors ..... 218
Figure V-6. Retroviral Math5 overexpression does not stimulate RGC fate
or cell cycle exit in retinal explant cultures .................................................... 219
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Figure VI-S1. ONA Patient 1 carries a duplication that disrupts CNTN4 ...... 278
Figure VI-S2. The chromosome 3p26 duplication in Patient 1 is capable of
producing a truncated CNTN4 mRNA ........................................................... 279
Figure VI-S3. Chromosome 14q23 deletion in optic nerve aplasia
Patient 2 encompasses the OTX2 gene ........................................................ 281
Figure VI-S4. ATOH7 variants have similar protein stability ......................... 282
Figure VI-S5. Low power views of retinal explant rescue experiments ......... 283
CHAPTER VII:
Figure VII-1. Outline of the Mosaic Analysis of Double Markers (MADM)
strategy ......................................................................................................... 297
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LIST OF TABLES
CHAPTER II:
Table II-S1. Oligonucleotide primers in this study .......................................... 76
Table II-S2. PCR conditions in this study ....................................................... 77
Table II-S3. DNA sequence flanking deletions in RT-PCR products .............. 78
CHAPTER III:
Table III-1. Cell type distribution of Math5 lineage descendants in
wild-type Math5>Cre transgenic retinas ........................................................ 139
Table III-S1. Cell type distribution of Math5 lineage descendants in Math5
mutant mice ................................................................................................... 143
Table III-S2. Dual reporter concordance for Math5>Cre labeled
retinal cells .................................................................................................... 144
Table III-S3. Cumulative BrdU labeling experiment (E10.5 to P0) ................ 145
Table III-S4. Birthdates of Math5 lineage retinal descendants ..................... 146
CHAPTER V:
Table V-S1. Oligonucleotide primers and PCR conditions used
in this study. ................................................................................................... 239
CHAPTER VI:
Table VI-S1. Clinical Features of optic nerve aplasia cases......................... 284
Table VI-S2. Oligonucleotide primers and PCR conditions used
in this study ................................................................................................... 285
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ABSTRACT
Vertebrate retinal histogenesis is controlled by both intrinsic transcriptional
programs and the microenvironment. The basic helix-loop-helix (bHLH) factor
Math5 (Atoh7) is required for differentiation of retinal ganglion cells (RGC), which
form the optic nerve. Math5 knockout mice lack RGCs, but only 10% of Math5expressing progenitors adopt the RGC fate, and only 55% of RGCs are lineal
descendents of Math5+ cells.
To define the role of Math5 in RGC development, I characterized the
transcriptional anatomy of mouse Math5, and showed that it is an unspliced,
single-exon gene, contrary to a recent high-profile report. I then tested the
contribution of Math5-expressing cells to the earliest born cohort of mouse retinal
neurons, which consist primarily of RGCs (~80%). Unexpectedly, I found that
only 20-30% of this cohort expresses Math5, yet most early RGCs depend on
Math5 function, suggesting a non-autonomous role for Math5-expressing cells in
RGC specification.
Next, I evaluated the onset of Math5 expression, and that of RGC markers
Brn3b and Isl1, with respect to the terminal cell cycle. Surprisingly, these
markers were expressed by neurogenic cells prior to terminal mitosis during early
development (<E14), but restricted to post-mitotic cells during later stages. By
retroviral clone analysis, I confirmed that early neurogenic cells often divide
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CHAPTER I
VERTEBRATE RETINAL DEVELOPMENT
limiting membrane (OLM) to the inner limiting membrane (ILM) (Fig. I-1B). These
glial cells are important for maintaining the physiology and laminar structure of
the retina (Bringmann et al., 2006; Willbold et al., 2000).
Bipolar cells synapse on retinal ganglion cells (RGCs), whose cell bodies
reside in the GCL. RGC axons coalesce to form the optic nerves, which project
to areas in the central nervous system (CNS) that are responsible for visual
processing, and autonomic tasks, including eye positioning and object tracking,
modulating sensitivity to different levels of light, and controlling circadian rhythm
(Rodieck, 1998). These areas include the superior colliculi (SC), the lateral
geniculate nuclei (LGN), intergeniculate leaflets, the pretectum, the accessory
optic system (AOS), and the suprachiasmatic nuclei (SCN) (Schiller and Malpeli,
1977; Simpson, 1984). A small number of RGCs have intrinsic photosensitivity
(ipRGCs), express the photopigment melanopsin, project to the SCN, and are
important for photoentrainment of circadian rhythms (Gooley et al., 2001; Hattar
et al., 2002; Provencio et al., 1998). In addition to RGCs, the mammalian GCL
contains displaced amacrine cells (Masland, 1988), which have similar functions
as their INL counterparts.
There is significant diversity in the retina beyond the seven major cell
types (Masland, 2001), with particularly many subclasses of amacrine (MacNeil
and Masland, 1998), bipolar (Ghosh et al., 2004; Kim et al., 2008) and ganglion
cell (Rockhill et al., 2002) neurons. The relative abundance of each retinal cell
type is also quite different (Jeon et al., 1998). In particular, the ratio between
photoreceptors (input) and RGCs (output) highlights the importance of signal
integration, and varies widely across the retina, giving different levels of spatial
resolution. The overall number of photoreceptors vastly exceeds the number of
bipolar cells (~11:1), which in turn exceeds the number of ganglion cells (~12:1).
This integration greatly increases the sensitivity of the retina.
Young, 1985a). This temporal profile overlaps significantly with that of cone,
horizontal, amacrine cells. Rods, Mller glia and bipolar cells have
characteristically later birthdates, which peak in the perinatal or neonatal periods
in rodents.
Clonal analyses of retinal progenitors in frog and rodents, using retroviral
or plasmid vectors with histochemical markers, have revealed that the seven
major retinal cell types are generated from a common progenitor pool (Holt et al.,
1988; Turner and Cepko, 1987; Turner et al., 1990; Wetts and Fraser, 1988). In
these studies, composition and size among various clones was largely
heterogeneous, suggesting that no strictly determined lineages exist in the retina,
unlike those observed in the C. elegans nervous system (Ruvkun, 1997).
Indeed, large clones could contain both early and late cell types. Additionally,
the existence of two-cell clones with discordant fates suggests that commitment
to a particular cell fate must occur during or after the terminal division.
Heterochronic co-culture and transplantation experiments in rodents
likewise provided clues towards the mechanism of retinal fate determination.
When late (post-natal) progenitors were mixed at low density with a large
number of early cells (early embryonic), these late progenitors were incapable
of adopting early fates, suggesting an intrinsic restriction in competence, i.e. the
ability to respond to environmental signals (Belliveau and Cepko, 1999; Reh,
1992; Watanabe and Raff, 1990). In reciprocal experiments, early progenitors
were capable of adopting late fates, such as rod photoreceptor fate (Reh, 1992;
Watanabe and Raff, 1990). However, these RPCs were still heavily biased
apical (sclerad) surface of the retina, and return to the base in G1. After terminal
M phase, differentiating neurons migrate to their final laminar positions in the
neuroepithelium.
The dynamics of the RPC cell cycle have been extensively characterized
in rodents and other vertebrate species by window and cumulative nucleoside
labeling, cell counting, and percent labeled mitosis (PLM) methods (Alexiades
and Cepko, 1996; Fujita, 1962; Li et al., 2000; Sinitsina, 1971; Young, 1985b).
The common conclusion from these studies is that the overall cell cycle
progressively lengthens during development. This increase is largely attributed
to prolonged G1 and S phases of the cell cycle (Alexiades and Cepko, 1996;
Young, 1985b). Though much is known about the kinetics of the RPC cell cycle,
it remains unclear what role cycle dynamics play in the ultimate cell fate choice.
It is clear, however, that the cell cycle exit and fate determination are
correlated. Genetic overexpression or loss of cell cycle regulators, such as p27,
p57, Rb, cyclinD1, can have wide-spread effects on the retina, including the
distribution of retinal cell types (Das et al., 2009; Dyer and Cepko, 2000; Fantl et
al., 1995; Ohnuma et al., 1999; Zhang et al., 2004). Mitogenic factors, such as
Notch or Shh signaling molecules, tend to promote generation of late fates
(Jadhav et al., 2006; Levine et al., 1997); while loss of these and the presence of
neurogenic factors promote the generation of early fates (Dyer et al., 2003;
Riesenberg et al., 2009; Yaron et al., 2006). Despite these large influences, cell
cycle progression is not required for the generation of a wide array of neuronal
cell types in frog retina (Harris and Hartenstein, 1991). Thus, it remains to be
that 50-70% of RGCs undergo cell death, with peak apoptosis in the neonatal
period (Crespo et al., 1985; Erkman et al., 2000; Farah and Easter, 2005; GalliResta and Ensini, 1996; Strom and Williams, 1998; Young, 1984). Activitydependent axon competition and defects in pathfinding are thought to be the
major mechanism underlying this neonatal cell death (Fawcett et al., 1984;
O'Leary et al., 1986; Scheetz et al., 1995).
10
Delta ligands and secreted Shh, which are produced by nascent RGCs, are
known to negatively RGC genesis (Austin et al., 1995; Belliveau and Cepko,
1999; Waid and McLoon, 1998; Wang et al., 2005; Zhang and Yang, 2001).
These same signaling pathways (Notch and Hedgehog) also promote the
generation of other cell types (Furukawa et al., 2000; Jadhav et al., 2006; Levine
et al., 1997; Shkumatava et al., 2004). It is unclear, however, whether these
effects on cell fate are secondary to altering general progenitor dynamics (Austin
et al., 1995; Jensen and Wallace, 1997; Levine et al., 1997). For example, the
mitogenic and fate effects of Notch signaling are intertwined. Inhibition of the
Notch pathway may promote early fates simply by causing premature cell cycle
exit, increasing the fraction of cells that are specified in an early environment
(Austin et al., 1995; Nelson et al., 2007). Additionally, extrinsic factors can have
dual roles. They can support the differentiation of a particular cell type, while
inhibiting specification of new cells. For example, Shh produced in RGCs can
also promote axon pathfinding among nascent ganglion cells (Kolpak et al.,
2005; Sanchez-Camacho and Bovolenta, 2008). A vast array of other signaling
molecules, including ciliary neurotrophic factor (CNTF), fibroblast growth factor
(FGF), and transforming growth factor (TGF), also actively fine tune the
production of different cell types in the retina (Cepko, 1999; Ezzeddine et al.,
1997; Kim et al., 2005; Yang, 2004).
In an integrated view of retinal histogenesis, cell fate is fundamentally
controlled by intrinsic transcriptional cascades, which are influenced by the
environment (Fig. I-2D). A critical point in a progenitors life history is the
11
decision to exit the cell cycle. Thus, several core homeodomain (HD) and basichelix-loop-helix (bHLH) transcription factors, such as Chx10, Pax6, and Hes1,
are thought to control the progenitor proliferation and maintain RPCs in an
undifferentiated state (Ashery-Padan and Gruss, 2001; Marquardt, 2003;
Takatsuka et al., 2004).
Histotypic differentiation, the acquisition of unique cellular morphology and
functional features, is controlled by downstream transcriptional hierarchies, which
are similarly complex (Fig. I-2D). Although precise control of specification
remains largely unclear, many insights have emerged in recent years regarding
the hierarchy of differentiation from analysis of gene expression in loss- and gainof-function animal models. For example, in the development of inhibitory
amacrine and horizontal cells, it clear that both the HD factor FoxN4 and bHLH
factor Ptf1a (P48) are necessary for the development of these cell types, and
also bias progenitors toward this fate (Fujitani et al., 2006; Li et al., 2004). The
pattern of molecular epistasis (Lehner, 2011) in reciprocal mutants suggests that
FoxN4 precedes Ptf1a in the pathway. Loss of Ptf1a does not affect FoxN4
expression, but loss of FoxN4 impairs Ptf1a expression. Thus, a clear hierarchy
emerges, in which FoxN4 functions upstream of Ptf1a (Fujitani et al., 2006).
Similar transcriptional hierarchies have been established for the differentiation of
photoreceptors and other cell types (Ohsawa and Kageyama, 2008; Swaroop et
al., 2010). However, basic mechanisms controlling the fate choice, i.e. the
events that trigger these cascades, remain largely unknown.
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al., 2003; Wee et al., 2002). Because the inner retina is thin, and bipolar neurons
are deficient, the mutants have abnormal retinal electrophysiology, with reduced
b-wave amplitudes in electroretinogram (ERG) recordings (Brzezinski et al.,
2005). Math5 -/- mice retain each of the other 7 major cell types, indicating that
Math5 is only required for ganglion cell development. Similarly, in zebrafish, the
ortholog ath5 is required for RGC development, as a point mutation in this gene
(lakritz) causes agenesis of RGCs and optic nerves (Kay et al., 2001).
Although Math5 is required for RGC development, it acts as a permissive
factor for RGC genesis. Lineage-tracing studies using a Math5-Cre knock-in
allele and BAC transgene have shown that Math5 descendents comprise most
major cell types in addition to RGCs (Brzezinski and Glaser, 2004; Feng et al.,
2010; Yang et al., 2003). Likewise, Math5 over-expression studies in chick and
frog suggest that this factor is not instructive for RGC fate. In both species,
overexpression of orthologs Xath5 and Cath5 in proliferating RPCs during early
development mildly biases progenitors toward the RGC fate (Brown et al., 1998;
Kanekar et al., 1997; Liu et al., 2001). However, when Xath5 is misexpressed
during late developmental stages in frogs, it promotes other neuronal fates
(Moore et al., 2002). In general, high-level expression of proneural bHLH factors
may drive cell cycle exit and neuronal differentiation (Farah et al., 2000). Thus,
the observed shifts in fate distribution may largely be due to this property of
bHLH factors, rather than a specific role of ath5.
The hierarchy of RGC differentiation remains incompletely understood
despite many genetic and genomic studies (Mu and Klein, 2004). It is clear that
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retina is supplied by the hyaloid artery, which enters the eye through the optic
stalk and the embryonic fissure during early development (Zhu et al., 1999). The
choroidal and hyaloid vessels branch from the dorsal ophthalmic artery. The
hyaloid artery extends into the vitreous and forms the hyaloid vascular network,
which nourishes the lens and the inner retina (Fig. I-4). In order to accommodate
vision through an optically clear media, this vasculature remodels during the
early post-natal period in mice or late gestation in humans (Fruttiger, 2007;
Gariano and Gardner, 2005). The hyaloid vasculature regresses by apoptosis of
vascular endothelial cells, which is triggered by macrophage phagocytosis (Lang
et al., 1994). In turn, the blood vessels sprout by angiogenesis, following a
migrating astrocyte network that initiates at the optic stalk and spreads radially
along the inner surface of the retina (Fruttiger, 2007). These surface vessels
then dive into the retina to form two deep vascular plexi that surround the INL
and supply the inner retina. Astrocytes, marked by platelet-derived growth factor
receptor alpha (PDGFR) expression, form a scaffold for angiogenesis and are
important for proper development of the vasculature (Dorrell et al., 2002; Laterra
et al., 1990; Watanabe and Raff, 1988). RGCs elaborate platelet-derived growth
factor A ligand (PDGFRA), sonic hedgehog (Shh), and other trophic molecules
that are critical for astrocyte proliferation and migration (Dakubo et al., 2003;
Fruttiger et al., 1996; Fruttiger et al., 2000).
The retinal vasculature develops abnormally in Atoh7 mutant mice lacking
RGCs (Brzezinski et al., 2003; Edwards et al., 2011) (Fig. I-4). In these mice, the
mature retinal vasculature fails to form and the hyaloid vasculature persists,
16
continuing to supply the mature retina. These abnormal vessels typically invade
and neovascularize the neural retina, and they are prone to hemorrhage, which
can occur in the subretinal space, vitreous, or spread into the anterior chamber
(Brown et al., 2001; Brzezinski et al., 2003).
Diseases of the optic nerve and retinal vasculature
Congenital optic nerve diseases are important causes of hereditary
blindness (Taylor, 2007). Optic nerve hypoplasia (ONH), in which nerves are
reduced in size, and optic nerve aplasia (ONA), in which nerves are completely
absent, are thought to be caused by primary defects in the specification,
differentiation, or survival of RGCs (Lambert et al., 1987). The less severe
clinical entity, ONH, is much more common than ONA (Borchert and GarciaFilion, 2008). Each can occur as an isolated malformation, or as part of a
syndrome, together with central nervous system (CNS) and pituitary defects
(Borchert and Garcia-Filion, 2008; McCabe et al., 2011). In ONA, the mature
retinal vasculature typically fails to develop, and fetal vessels may persist in the
vitreous (Blanco et al., 1992; Brodsky et al., 2004; Lee et al., 1996; Little et al.,
1976; Scott et al., 1997). Most cases of ONA and ONH are sporadic. Few
genetic causes have been identified and these explain only a very small fraction
of cases. Mutations in HESX1, OTX2, SOX2 or PAX6 can lead to severe ONH
or ONA (Azuma et al., 2003; Dattani et al., 1998; Kelberman and Dattani, 2007;
McCabe et al., 2011; Ragge et al., 2005). These Mendelian cases usually
present with hypothalamic-pituitary dysfunction, and global eye and CNS
malformations. The diagnostic terms Syndrome of Optic Nerve Hypoplasia
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been noted in some patients with normal vision (Copt et al., 1999). Despite
numerous advances in recent years, many cases of glaucoma remain
unexplained, and the pathogenesis is incompletely understood.
Retinal vascular diseases are also major causes of blindness (Gariano
and Gardner, 2005). Retinopathy of prematurity (ROP) is vascular disorder that
primarily affects premature infants and can be triggered by excessive oxygen
exposure in newborns. It is characterized by vascular loss and incomplete
vascularization followed by a compensatory hyperproliferation of the remaining
retinal vessels (Chen and Smith, 2007). ROP can be accompanied by persistent
hyperplasia of the primary vitreous (PHPV). In this disorder, the primary fetal
vasculature fail to regress, likely due to impaired apoptosis, abnormalities in the
mature vascular development, or hypoxic conditions (Goldberg, 1997; Reese,
1955; Shastry, 2009). Most cases are sporadic, but rare familial cases have
been characterized (Haddad et al., 1978; Khaliq et al., 2001). PHPV predisposes
retinas towards detachment, hemorrhage, and anterior chamber defects (Pruett,
1975). A small number of cases may be explained by genetic defects in the Wnt
signaling pathway (Robitaille et al., 2009). Mutations in these genes can also
cause Norries disease or familial exudative vitreoretinopathy (FEVR), which
share similar clinical features (Berger et al., 1992a; Berger et al., 1992b; Gariano
and Gardner, 2005; Robitaille et al., 2009). Few other genetic causes for these
diseases have been identified.
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Novel insights into RGC development and the role of Math5 (ATOH7)
Ganglion cells play an essential role in the visual system. They transmit
all visual information to the CNS and are required for the development of retinal
vasculature. Thus, the development of RGCs remains an important area of study
for understanding the causes of blindness and the mechanisms of visual system
development. In this dissertation, I have focused my analysis on the ATOH7
(Math5) gene, which plays a particularly important role in the initial development
of this retinal cell type.
In Chapter II, I thoroughly and systematically analyze the transcriptional
architecture of the mouse Math5 gene. I found that this single-exon gene makes
one predominant mRNA isoform, which is unspliced. This study starkly contrasts
a prior report that suggested a major role of alternative splicing in the regulation
of the gene (Kanadia and Cepko, 2010).
In Chapter III, Joseph Brzezinski and I quantitatively characterized the
lineage and cell cycle properties of the Math5-expressing cells. We show that
Math5 is variably expressed in progenitors during or after their terminal cell cycle,
with a gradual restriction to post-mitotic cells as development proceeds. Using
Math5>Cre BAC transgenic mice, we show that only 11% of lineage-derived cells
adopt the RGC fate, and that only 55% of ganglion cells are marked by Math5
expression. Surprisingly, the Math5-independent fraction is substantial even
during early development, suggesting a non-autonomous role of Math5 in
ganglion cell fate specification.
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Figure I-1. Structure of the eye and retina. (A) Basic fuschin and methylene
blue staining of a central section of a mouse eye. Major structures of the eye
are highlighted. (B) H+E staining showing the trilaminated mouse retina. A
schematic diagram of the 7 major retinal cell types is shown on the right. rpe,
retinal pigmented epithelium; on, optic nerve; ret, retina; cb, ciliary body; corn,
cornea; onl, outer nuclear layer, inl, inner nuclear layer; gcl, ganglion cell
layer; ilm, inner limiting membrane; olm, outer limiting membrane; r, rod; c,
cone; hz, horizontal neuron; bip, bipolar cell; am, amacrine; da, displaced
amacrine; rgc, retinal ganglion cell; mg, Mller glia.
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Figure I-3. Math5 (Atoh7) knockout mice have no optic nerves, and
severe deficiencies in RGCs. (A-B) Views of the brain of wild-type (A)
and Math5 -/- mice (B). The optic chiasm and nerves are absent in
Math5 -/- mice. (C-D) Hematoxylin and eosin staining of retinal sections
from wild-type (C) and Math5 -/- (D) mice. Math5 mutant retinas are
thinner than wild-type, and have a hypocellular ganglion cell layer that is
composed of displaced amacrine cells. Adapted from Brown et al., 2001.
onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer.
26
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CHAPTER II
A CRITICAL ANALYSIS OF MATH5 (ATOH7) mRNA SPLICING IN THE
DEVELOPING MOUSE RETINA
Abstract
The Math5 (Atoh7) gene is transiently expressed during retinogenesis by
progenitors exiting mitosis, and is essential for ganglion cell (RGC) development.
Math5 contains a single exon, and its 1.7 kb mRNA encodes a 149-aa
polypeptide (Brown et al. 1998). Mouse Math5 mutants have essentially no
RGCs or optic nerves. Given the importance of this gene in retinal development,
we thoroughly investigated the possibility of Math5 mRNA splicing by Northern
blot, 3 RACE, RNase protection assays, and RT-PCR, using RNAs extracted
from embryonic eyes and adult cerebellum, or transcribed in vitro from cDNA
clones. Because Math5 mRNA contains an elevated G+C content, we used
graded concentrations of betaine, an isostabilizing agent that disrupts secondary
structure. Although ~10% of cerebellar Math5 RNAs are spliced, truncating the
polypeptide, our results show few, if any, spliced Math5 transcripts exist in the
developing retina (<1%). Rare deleted cDNAs do arise via RT-mediated RNA
template switching in vitro, and are selectively amplified during PCR. These data
differ starkly from a recent study (Kanadia and Cepko 2010), which concluded
that the vast majority of Math5 and other bHLH transcripts are spliced to
29
generate noncoding RNAs. Our findings clarify the architecture of the Math5
gene and its mechanism of action.
Introduction
The vertebrate retina develops from a single multipotent progenitor
population, which gives rise to seven major cell types rod and cone
photoreceptors; amacrine, bipolar and horizontal interneurons; Muller glia; and
retinal ganglion cells (RGCs) (Holt et al., 1988; Turner and Cepko, 1987). These
diverse cell types emerge from the mitotic progenitor pool in rough sequential
order, with overlapping birthdates (Livesey and Cepko, 2001; Wong and
Rapaport, 2009). RGCs are the first-born retinal cell type in every vertebrate
examined (Altschuler et al., 1991). These cells transmit all visual information from
the eye to the brain, via their axons, which comprise the optic nerves. The gene
network regulating retinogenesis is an active area of investigation.
An important clue toward understanding the mechanism of vertebrate
retinal fate specification was the discovery of Math5 (Atoh7), a proneural basicloop-helix (bHLH) transcription factor that is evolutionarily related to Drosophila
Atonal and mouse Math1 (Atoh1) (Brown et al., 1998; Kanekar et al., 1997). The
mouse Math5 gene is expressed transiently in retinal cells exiting mitosis, from
E11.5 until P0, in a pattern that is correlated with the onset of neurogenesis, and
it is necessary for RGC fate specification. Math5 mutant mice lack RGCs and
optic nerves (Brown et al., 2001; Wang et al., 2001), and have secondary defects
in retinal vascularization (Brzezinski et al., 2003) and circadian photoentrainment
30
31
32
33
34
35
were matched for length and G+C content. Products were diluted to 1:50 to 1:200
in formamide and co-electrophoresed with GS-600 LIZ size marker in a 3730XL
capillary DNA Analyzer (Applied Biosystems). The fluorescence intensity of each
amplimer and the ratio of spliced to unspliced PCR products were calculated
using GeneMarker (SoftGenetics), from the sum of major peaks in triplicate
experiments.
Rapid amplification of cDNA ends (3 RACE)
First-strand cDNA synthesis was performed from retinal RNA as described
above, using 10 pmol adapter primer (AP, Table II-S1). One L of the cDNA
reaction was then used to amplify 3 terminal sequences using primers and
conditions in Tables II-S1 and II-S2. To minimize spurious products from
unrelated genes, a second round of PCR was performed using nested primers,
following a conventional nested 3 RACE strategy (Frohman, 1993).
RNase protection assays (RPA)
RNase protection assays (Melton et al., 1984) were conducted using the RP-III
kit (Ambion). Antisense cRNA probes were transcribed from PCR products A
and B (Fig. II-5) cloned in pCR4-TOPO. One g of each plasmid was digested
with NotI and transcribed with T3 RNA polymerase as described above, except
that 125 pmol (75 Ci) or 113 pmol (90 Ci) 32P-[]-CTP was included in probe A
and B reactions, respectively, with 200 pmol CTP and 10 nmol of ATP, GTP and
TTP. This yielded 366 and 632 nt cRNA products with 301 nt (A) and 567 nt (B)
direct sequence homology to Math5. Probes were purified by electrophoresis
36
through denaturing 6% polyacrylamide gels and eluted for 3-4 hrs at 37C. Ten
g of DNaseI-treated E14.5 eye RNA, yeast RNA (Ambion), or yeast RNA spiked
with 10 ng Math5 IVT product (ECO or FL) was precipitated in 2.5 M ammonium
acetate 70% ethanol and resuspended in 8 l hybridization buffer. The RNAs
were hybridized with 2 l probe A (8 x 104 cpm) or probe B (1.2 x 105 cpm) for
13 hr at 42C, and digested with RNase A+T1 (1:100) for 30min at 37C, and
co-precipitated with glycogen and 5 g yeast carrier RNA. Reactions were
electrophoresed through 6% polyacrylamide denaturing gels (0.4 mm) in 6 M
Urea and 0.5X TBE. dsDNA size markers were prepared by radiolabeling MspIdigested pBR322 with 32P-[]-dCTP and Klenow DNA polymerase. The dried
gels were exposed to phosphor screens for 12-14 hrs and imaged using a
Typhoon scanner (Molecular Dynamics) at 0.2 mm resolution. Yeast RNA
controls were included RNase, to assess the probe integrity and the
completeness of digestion.
Informatics
Sequence alignments, G+C and antigenicity profiles, and PCR primer
optimization were performed using MacVector (Accelrys) software and NCBI
BLAST servers. Math5 polyadenylation sites were predicted using the polyADQ
(Tabaska and Zhang, 1999) web server (rulai.cshl.org/tools/polyadq/). Scores
were calculated for a 6.0 kb sequence extending from the transcription start site
(Fig. II-1D), using default threshold values. RNA secondary structures were
predicted by free-energy minimization (Mathews et al., 1999; Zuker, 2003) using
the M-fold web server (mfold.bioinfo.rpi.edu/). Expressed sequence tags (ESTs)
37
for mouse and human bHLH cDNAs were accessed through the UCSC genome
browser (genome.ucsc.edu/).
Math5 antibodies
Commercial and custom antibodies to Math5 peptides and recombinant proteins
are indicated in Fig. II-S2 along with the immunogen, including the Abcam
polyclonal reagent (ab13536) cited by Kanadia and Cepko (2010). Custom rabbit
polyclonal sera were generated using internal (RCEQRGRDHP) or C-terminal
(RLFGFQPEPFPMAS) Math5 peptide haptens coupled to KLH (keyhole limpet
hemocyanin) via a cysteine thiol linkage (Research Genetics, Huntsville, AL), and
were affinity purified.
Cell transfection and Western analysis
NIH 3T3 fibroblast cultures were transfected with expression plasmid DNA (1 g
per 60 mm plate) for native or N-terminal 6x Myc-tagged versions of mouse or
human ATOH7 proteins, or empty vector, using Fugene-6 reagent (Roche), with
0.1 g pUS2-EGFP as an internal control. These plasmids were prepared by
inserting ATOH7 coding regions from genomic phage, plasmid or BAC clones
into pCS2 and pCS2MT vectors (Rupp et al., 1994) and verifying the sequence.
Mouse pCS2-Math5 and pCS2MT-Math5 plasmids were described previously
(Brown et al., 1998). After 48 hrs, cells were harvested in PBS with protease
inhibitors (CompleteTM, Roche), lysed in RIPA buffer (Harlow and Lane, 1988),
sonicated, and centrifuged at 13,000 g for 15 min at 4C. Soluble proteins were
electrophoresed through NuPAGE Novex Bis-Tris 4-12 % polyacrylamide gels
38
Results
40
particularly in the 0.8-1.0 kb size range expected for spliced isoforms lacking the
coding region. This pattern resembles Northern data obtained by Kanadia and
Cepko with UTR probes (cf. Fig.1f and 1f), but appears inverted compared to the
unsized blot hybridized with a CDS probe in their report (cf. Fig. 1f). We cannot
explain this discrepancy.
To confirm our identification of the major Math5 polyadenylation site
(Brown et al., 1998) and define the 3 terminus of the longer, 4.4 kb transcript, we
first surveyed the 3 Math5 genomic region for favorable pA signals using the
polyADQ weighted statistical algorithm (Tabaska and Zhang, 1999). Among eight
potential pA sites downstream from the transcription start site (TSS), two had
significant polyADQ scores (nos.1 and 6, Fig. II-2B,C), and these were consistent
with the observed transcript sizes. We then looked for mRNAs terminating at pA1
and pA6 in parallel 3 RACE experiments (Frohman, 1993), using E14.5 total eye
RNA and nested primers positioned upstream of each site (Figure 2b,d). From
the size and sequence of the products (Fig. II-2D-F), and our Northern data, we
conclude that there are two principal Math5 transcripts in the retina, 1.7 kb and
4.4 kb in length, and that both of these transcripts are unspliced. This
interpretation is further supported by the curation of additional mouse cDNAs,
represented as 56 expressed sequenced tags (ESTs) and two Genbank cDNAs
in the NCBI database (Fig. II-S1). Only two ESTs and one cDNA, originating from
the adult cerebellum, appear to be authentic splice products (see below), and
these do not correspond to the retinal isoforms reported by Kanadia and Cepko
(2010).
41
In addition to the coding region, Math5 mRNA has three notable features
relevant to this study (Fig. II-1A). First, the 5 half is highly enriched in G+C
nucleotides (Fig. II-1B), with >85% G+C content in the 150 nt segment spanning
codons 7 to 57. Math5 mRNA thus has the potential to form compact,
thermodynamically stable secondary structures, owing to the third hydrogen bond
in G-C pairs compared to A-U pairs, and the ability of guanine residues to
interact with uracil in folded RNA (Mathews et al., 1999). The elevated G+C
content is also predicted to affect folding of the (+) and (-) strand cDNA
templates, compromising DNA polymerase processivity. Second, the 5 segment
of the gene is enriched for specific trinucleotide elements (Py-G-C) that are
known to cause DNA polymerase pausing (Mytelka and Chamberlin, 1996) (Fig.
II-1C). These account for 15.7% of the trinucleotides in this segment (47 of 300,
for both DNA strands), which is 1.73 fold higher than expected from
mononucleotide frequencies. Third, mouse Math5 mRNA contains 30-nucleotide
imperfect direct repeats (DRs), located in the 5 and 3 UTRs (Fig. II-1A,E).
These UTR repeats are not conserved among mammalian ATOH7 mRNAs.
Sensitivity of Math5 PCR to template folding in vitro
Our Northern analysis, screening of cDNA libraries, and analysis of ESTs
contrasts starkly with the abundant, heterogeneous splicing recently reported for
the Math5 gene (Kanadia and Cepko, 2010). As a first step to resolve this
difference, we performed a series of RT-PCR experiments using the same
primers (LP8 and LP4, Fig. II-1A and Table II-S1) and similar conditions (Table IIS2) as these authors. Using a thermostable reverse transcriptase (RT)
42
43
44
segment (Fig. II-3C,D). In the absence of betaine, these primers did not amplify
any product. However, when 2-3X MasterampTM was included in the PCR, only
the expected 486 bp amplimer was observed. When we extended the PCR
beyond 35 cycles, preincubated the reaction at 25C (cold start) or used crude
Taq polymerase preparations in the absence of betaine, a heterogeneous group
of deleted (lacunar) products was observed (not shown), with a size and
sequence distribution (Fig. II-4D, Table II-S3) similar to that reported by Kanadia
and Cepko.
45
46
47
48
49
The reactions involved the terminal portion of the Math5 coding region and
3 UTR, and the products were similar in size (301 vs. 228 bp) and G+C content
(47.8 vs. 52.2 %). In contrast to the embryonic retina, we observed a moderate
level of alternative mRNA splicing in the adult cerebellum, involving 11 2 % of
Math5 transcripts. The major (1.7 kb) and minor (1.5 kb) cerebellar splice forms
were not previously resolved in Northern blots (Saul et al., 2008), presumably
because of the difference in abundance, and the effect of polyA tail heterogeneity
(with an expected mean length of 250 adenosines, (Wahle, 1995). This shorter
isoform was not detected in the embryonic retina (Fig. II-S3D).
Discussion
We have critically defined the transcriptional anatomy of the Math5 gene,
and characterized alternatively spliced mRNAs. In contrast to the adult
cerebellum, Math5 mRNA is not significantly spliced in the developing retina.
This conclusion is supported by six independent lines of evidence: Northern
analysis; RT-PCR analysis of natural and IVT-derived RNAs in the presence of
graded betaine concentrations; triplex RT-PCR analysis; EST data; and
ribonuclease protection assays. Our findings differ sharply from the recent report
of Kanadia and Cepko (2010). Three major factors contribute to the technical
artifacts observed by these authors: [1] intense secondary structure in the >85%
GC-rich segment of Math5 RNA and cDNA, which blocks the progression of
polymerase enzymes, creating a powerful negative selection; [2] RT template
switching in vitro; and [3] the existence of a vanishingly small population of
50
aberrantly spliced Math5 mRNAs (Fig. II-7A). In view of these results, further
investigation of Ngn3 splicing may be warranted (Fig. II-S4).
The GC-rich coding segment of Math5 (Fig. II-1B) evidently forms a
Gordian knot of secondary structure (Fig. II-7B,C), so dense that it favors the
amplification of minor cDNA products, representing less than 1% of Math5
molecules. G+C sequence bias is a well known problem in cDNA profiling studies
(Blackshaw et al., 2004; Margulies et al., 2001). The folded hairpin structure of
Math5 mRNA is relaxed in the presence of betaine. In vivo, local melting is
presumably catalyzed by DNA- and RNA-binding proteins, allowing Math5
replication, transcription and translation. However, the tight RNA secondary
structure may have consequences for Math5 protein expression. For example,
translation may require specific mRNA unwinding activity, creating another
potential mode of post-transcriptional regulation (Gray and Hentze, 1994).
Indeed, mRNA hairpins are known to impede ribosome elongation (Baim et al.,
1985) and G+C content is inversely correlated with translation efficiency
(Kenneson et al., 2001). If translation of the GC-rich Math5 mRNA were
hypersensitive to ribosome functional status, this may contribute to the disruption
of RGC development in Bst/+ mice, which have a mutation in the Rpl24
riboprotein gene and severe optic nerve hypoplasia (Oliver et al., 2004).
On the basis of these results, we believe that the most likely explanation
for the plethora of deleted Math5 cDNAs (Fig. II-4) is RNA template-switching
during the reverse transcriptase reaction, at points of sequence micro-homology
(Fig. II-7A, Table II-S3) (Brincat et al., 2002). Indeed, RT polymerases are
51
52
53
and statistical support for donor sites in the Math5 transcript was relatively low
(max FGA score = 0.26). Indeed, the mouse genome contains many more weak,
potential splice sites than are actually utilized in vivo.
Among the numerous Math5 species reported by Kanadia and Cepko,
only one PCR product, termed ECO, is compatible with mRNA splicing. On the
basis of our results, we believe this solitary cDNA is derived from an aberrantly
spliced transcript, which has escaped normal quality control. First, the RNA
encodes no protein and has no demonstrated function. In other contexts, long
ncRNAs such as Xist and Air, have been shown to have regulatory roles (Mercer
et al., 2009), and a small number of bifunctional mRNAs have alternate coding
and noncoding isoforms (Chooniedass-Kothari et al., 2004). Second, the ECO
isoform is very rare, representing less than 1% of Math5 mRNA, and is thus
unlikely to have a significant role in regulating Math5 function or modulating
retinal cell fate determination.
An intriguing result from our study is the discovery that 11% of mature
Math5 transcripts in the adult cerebellum are bona fide spliced mRNAs. These
are predicted to encode a shorter Math5 protein, which lacks 20 amino acids
from the C-terminus and may exhibit unique molecular properties (Fig. II-S3).
However, its function is not known, and Math5 mutants have no overt cerebellar
phenotype (Saul et al., 2008).
Despite the intriguing hypothesis advanced by Kanadia and Cepko, our
results show splicing of Math5 mRNA into noncoding isoforms does not occur in
the developing retina at levels greater than 1% of transcripts. Further studies are
54
needed to determine the exact mechanism of Math5 action, how progenitors are
transformed into neurons, and how noncoding RNAs, including microRNAs, may
regulate Math5 expression, RGC development, and the diversification of ganglion
cell subtypes.
Acknowledgements
The authors are grateful to John Moran and David Turner for helpful
suggestions; to Dellaney Rudolph, Tien Le, Susan Tarl and the UM sequencing
core for technical support; and to John Moran, David Turner, Miriam Meisler,
Doug Engel, Chris Chou, and Terri Grodzicker for careful reading of the
manuscript. The research was funded by NIH R01 grants to TG (EY14259) and
NLB (EY13612) and The Glaucoma Foundation (TG). LP was supported by NIH
T32 grants to the University of Michigan Medical Scientist (GM07863) and Vision
Research (EY13934) Training Programs.
55
Figure II-1. Anatomy of the Math5 transcription unit. (A) Gene map showing the
major 1489 nt mRNA species; coding region (red box) and UTRs; direct repeats
(DR); major polyA signal (pA) and internal A-rich segment (A14); cerebellarspecific intron (Cb); and PCR primers used in this study (dark red). LP15 spans
the Cb intron junction. (B) Plot showing elevated GC content (red) across the
Math5 coding region, compared to the average value (49.98%) for the mouse
transcriptome (green) (Stolting et al., 2009). The 150 nt segment with >85% GC
and the 536 nt fold encompassing the coding region are indicated (brackets). (C)
Concentration of polymerase-refractory YGC trinucleotides in the proximal coding
region (both strands). (D) Magnified view of the Math5 promoter showing the
TATAA box, transcription start site (TSS) and 5 termini of cDNA clones (Brown
et al., 2002; Brown et al., 1998). (E) Sequence of UTR direct repeats.
56
57
Figure II-2. Math5 messenger RNAs. (A) Northern blot probed with 1.2 kb Math5
(JN4C) and 1.1 kb -actin cDNAs. Two Math5 mRNAs are visible (left
arrowheads), but no hybridizing RNA species is present in the 0.8-1.0 kb size
range. The RNA size ladder cross-hybridized to vector DNA in the plasmid
probes. (B) Map of the 3 UTR and flanking genomic DNA (6 kb), showing eight
potential polyA signals ATTAAA (blue) and AATAAA (green); the internal A14
priming site in the UTR (pA); interspersed repeats (gray); and the nested
3 RACE primers (dark red) for pA1 and pA6 sites, which have the most favorable
sequence context. Clones JN2 and BC092234 terminate at pA1, whereas
cDNAs JN1, JN4 and JN6 terminate at pA (Brown et al., 2002; Brown et al.,
1998). pA2 marks an A-rich genomic site captured in the pA6 assay. (C)
polyADQ scores for all potential pA sites, calculated using human genome
parameters (Tabaska and Zhang, 1999). Only pA1 and pA6 have scores above
threshold. (D) Embryonic eye RT-PCRs with 260 bp and 365 bp 3 RACE
products (arrowheads) showing utilization of pA1 and pA6 sites. The 900 bp
product was primed from pA2 (open arrowhead). m, marker (1 kb-plus ladder);
RT, reverse transcriptase. (E) Sequence of pA1 RACE products originating from
the 1.7 kb Math5 mRNA. (F) Sequence of pA6 RACE products originating from
the 4.4 kb Math5 mRNA.
58
59
Figure II-3. Math5 embryonic eye RT-PCRs with increasing amounts of betaine. (A)
Agarose gel showing cDNA products amplified from DNase-treated E14.5 eye RNA
with UTR primers LP8 and LP4 in the presence of 0X, 1X, 2X and 3X MasterampTM.
When the betaine concentration was increased, only the full-length 1087bp Math5
cDNA product was visible; the 448bp ECO product was absent. No amplimers were
observed in the absence (-) of RNA template or RT enzyme. The identity of all PCR
products was verified by sequencing. (B) Similar PCR with a mouse genomic DNA
template, showing amplification of the identical full-length 1087bp product. (C,D)
Parallel PCRs were performed using internal primers LP6 and LP7. A single 486bp
Math5 product was amplified from cDNA or gDNA in 2-3XMasterampTM.
60
Figure II-4. RT-PCRs of Math5 RNA transcribed in vitro. (A) Diagram and
agarose gel showing linearized pJN4C and Math5 sense RNA generated by T3
polymerase and treated with DNaseI. (B) cDNA products amplified by RT-PCR
from IVT-derived RNA with UTR primers LP8 and LP4. Only the full-length
1087 bp Math5 cDNA product was amplified in the presence of 3X Masteramp
(MA, indicated above brackets). In the absence of betaine, a variety of weak
products were observed, with a heterogeneous deletion profile, reflecting a low
level of RT template switching. This background could be increased by using
suboptimal PCR conditions or omitting the mouse liver RNA carrier. IVT, in vitro
transcribed Math5 RNA (10 ng); ML, mouse liver RNA (3 g). (C) Similar
RT-PCRs performed using internal primers LP6 and LP7. Only the expected
486 bp cDNA was amplified in 3X MA, while spurious products were amplified at
lower MA concentrations. The right three panels in B and C represent adjacent
lanes in the same gels, displayed separately for clarity. (D) Alignment of lacunar
cDNAs generated from IVT or E14.5 eye RNA templates. The deletion profile is
comparable to the distribution reported by Kanadia and Cepko (2010, cf. Suppl.
Table 1 and Figure 1), using the same primer pairs with no precautions for GC
secondary structure. The sequence of breakpoints is given in Table S3, with
microhomology at the inferred sites of RT template switching.
61
62
Figure II-5. Triplex competitive RT-PCR assay to evaluate trace levels of Math5
splicing in the embryonic retina. (A) Diagram showing PCR strategy. The length
and % G+C of competing amplicons are comparable. (B) Agarose gel stained
with ethidium bromide, showing only the unspliced Math5 cDNA product in each
assay. (C). Capillary electrophoresis profiles showing triplex competitive
RT-PCR products (top panels) and the ECO product amplified with duplex UTR
primers in the presence of 1X MA (bottom panel). The common antisense primer
(LP4) was end-labeled with 6-FAM. From the peak areas measured in replicate
experiments and mixing controls, we estimate that the ECO product represents
0.4 to 1.0 percent of Math5 mRNA in the embryonic retina, which is near the
detection limit of this assay.
63
64
Figure II-6. Ribonuclease protection assays. (A) Diagram showing RPA strategy, with
Math5 cDNA, two different antisense cRNA probes, protected fragments expected for
FL (full length, unspliced) and ECO (spliced) transcripts, and positive control RNAs
generated by sense IVT reactions. (B) Autoradiogram, showing undigested probes A
and B (366 nt and 632 nt) and exclusively unspliced fragments protected by E14.5 eye
RNA (567 nt and 301 nt). No fragment corresponding to the presumptive ECO transcript
(212 nt) was protected by eye RNA using either cRNA probe, although a doublet of this
size was protected by the ECO IVT positive control. Background fragments observed
with probe B (arrowheads) are caused by intrinsic sensitivity of the cRNA-mRNA duplex
to RNase cleavage at particular sites and were also present in the full length IVT
positive control. The probe (no RNase) and IVT controls were diluted 20- and 10-fold
respectively, compared to the E14.5 eye RNA hybridization lanes.
65
66
Figure II-7. Model explaining the observed results. (A) Diagram showing the
likely origin of heterogeneous deleted Math5 cDNAs, through combined effects of
RT template switching, trace levels of aberrantly spliced ECO mRNA, and
powerful PCR selection favoring deletion of GC-rich coding sequences. (B)
Secondary structure predicted for the major 1489nt Math5 mRNA. This M-fold
circle diagram, generated by free energy (G) minimization, is magnified in Figure
S5. Red, blue and green arc lines indicate G-C, A-U and A-G base pairs. The
coding region, DRs and presumptive ECO splice sites are labeled. The 150nt
segment described in the text with >85% G+C, and the segment expanded in
panel C are marked. (C) Stem-loop diagram showing the 536nt fold that
encompasses the Math5 CDS with lowest free energy (G=-258 kcal/mol) and
Tm 82C. The major structural features in panels B and C are labeled alike. (D)
Junctional sequences for the ECO product with presumptive splice sites,
compared to the U1 consensus.
67
Figure II-S1. Math5 ESTs in the public domain. (A) Diagram modified from the
UCSC mouse genome browser (mm9 assembly, chr10 : 62,562,000 - 62,564,300)
showing 56 Math5 ESTs and 2 Genbank cDNAs (BC092234, AK005214), giving
a total n = 58, with 52 derived from the embryonic retina. Forty-three of these
retinal cDNAs cross the presumptive ECO junctions at the 5 or 3 side, and are
thus informative for splicing (83 %). Yet none originated from spliced mRNA. Of
the remaining six, from adult brain RNA (red), two cerebellar ESTs and one
cDNA were spliced at the Cb intron (yellow shading, see Figure S3). Nine 3
ESTs out of 21 terminate at pA1; the remaining 12 were primed from pA. (B)
Comparable region of the human genome (hg19 assembly,
chr10 : 69,992,300 - 69,990,000) showing one full-length Genbank cDNA and 7
unspliced ESTs.
68
69
Figure II-S2. Evaluation of Math5 antibodies. (A) Diagram of the mouse Math5
protein, showing the antigenic index (Jameson and Wolf, 1988) and positions of
immunogens used by various sources to prepare antibodies, as follows: a,b
internal and C-terminal peptides (Glaser lab); c, ab13536 (Abcam); d, AB5694
(Chemicon); e, EB07972 (Everest); f, 1A5 (multiple vendors). The immunogens
for D01P (Abnova) and MAb 1A5 were full-length or partial recombinant human
proteins (gray); all others were based on the mouse polypeptide (blue). No
immunogen was specified for ab78046 (Abcam). (B) Immunoblots of NIH3T3
cells co-transfected in parallel with pUS2-EGFP and pCS2 expression plasmids
for full-length mouse or human Math5 proteins six N-terminal Myc epitope tags,
or empty pCS2 vector. Five identical blots were probed using antibodies with
stated reactivity to mouse (ab13536, ab78046) or human (D01P) Math5; Myc or
GFP. The predicted mass for native and 6xMyc mouse Math5 proteins is 16.9
and 27.0 kDa, respectively. Antibody D01P detected the human polypeptides, but
not mouse. No other reagent tested was effective, including ab13536 (Abcam)
(Kanadia and Cepko, 2010), even when the Math5 proteins were massively
overexpressed. (C) Retinal sections from E15.5 embryos immunostained with
ab13536 sera. The immunofluorescence pattern was identical between wild-type
and Math5 -/- eyes and is thus nonspecific (Rhodes and Trimmer, 2006; Saper
and Sawchenko, 2003). This pattern, which includes lens and RPE nuclei, does
not fit the apical distribution of Math5 mRNA in the neuroblastic retina. The in situ
hybridization pattern of a Math5 cRNA probe spanning the 3 UTR and CDS
matches our previous reports (Brown et al., 1998; Hufnagel et al., 2010) and both
panels provided by Kanadia and Cepko (cf. Figure 1j and 1j).
70
71
72
73
Figure II-S4. Splicing patterns in the mouse Atonal-related bHLH genes. (A)
Phylogram of mouse Ato proteins, based on maximum parsimony analysis of the
bHLH domain across many taxa (Blackburn et al., 2008; Brown et al., 2002). (B)
Exon-intron organization of bHLH genes based on a survey of ESTs in the NCBI
database (Brett et al., 2002; Harrington et al., 2004). The eight mouse Ato
homologs either have unitary exon structures, or a single intron located in the
5'UTR. The Achaete-Scute homolog Mash1 (Ascl1) has a single intron in the
3'UTR. There is no obvious correlation between splicing patterns and locations in
the mouse genome. MMU, mouse chromo ESTs, number of expressed sequence
tags supporting the gene structure; *has minor alternative spliced product (Cb);
**has overlapping intergenic and antisense RNAs. The intron of one spliced
antisense EST (CF104925) for Ngn3 (Neurog3) overlaps the 5'UTR and coding
sequence of the sense strand. This antisense RNA is predicted to co-amplify in the
RT-PCR and may be mistaken for non-coding sense products.
74
Figure II-S5. Secondary structure for Math5 mRNA. This circle plot was
generated by free energy minimization of the 1489nt mRNA, and is enlarged
from Fig. II-7. Red, blue and green arc lines indicate G C, A U and A G base
pairs. The coding region, DRs and presumptive ECO splice sites are labeled.
The 150nt segment with >85% G+C, and 536nt segment spanning the CDS
are marked. The CDS contains a high density of G-C base pairs (red arcs),
which are deleted in rare, mis spliced RNAs.
75
76
S
AS
AS
AS
S
S
AS
S
AS
AS
AS
S
S
S
S
S
S
LP1
LP2
LP3
LP4
LP5
LP6
LP7
LP8
LP9
AP
UAP
LP10
LP11
LP12
LP13
LP14
LP15
TCTACTGCAAGCTGTCCAAACGCTC
AACATACAGGCTGTGTTGGTAGCTG
GGTAGCTGCTCAGAACATAAACAAGTCACAT
GTTTCTCCACCTCCTGAATGACGCT
GCCTCCCTATCTCCACTTCTCTTGTGT
GTGGATGAAGTCGGCCTGCAAA
TTTCTCCCCTAAGACCCAAATGGC
TCTCAGGCTTTCCCAGAGAACTGGA
TTTGCAGGCCGACTTCATCCAC
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT
GGCCACGCGTCGACTAGTAC
TCCCTATTGGGCGAAGTTGT
AGGGTGAAGTGCTTGCTGGT
GTTACAGGGCCTGCGAAATG
AAGCTGTCCAAGTACGAGACACTGC
CCTTTTCTGCTTAATTTCCTTCCCG
GGGTGCTAGGCTCCAG|GTTTC
Sequence (5 3)
RT-PCR
RT-PCR
RT-PCR
RT-PCR, triplex PCR
RT-PCR, 3RACE (pA1)
RT-PCR
RT-PCR
RT-PCR, triplex PCR
RT-PCR
3RACE
3RACE
3RACE (pA1)
3RACE (pA6)
3RACE (pA6)
RT-PCR, triplex PCR
triplex PCR
triplex PCR (Cb)*
Expt
1
2
3
4
5
6
7
1
2
Alternate names
Sup. Fig. 2
Sup. Table 2
Ori, orientation; S, sense; AS, antisense; AP, adapter primer; UAP, universal amplification primer
* primer sequence spans Cb intron junction
end-labeled with 6-carboxyfluorescein (6-FAM)
used by Kanadia and Cepko (2010)
Ori
Name
77
LP4*
LP4*
LP4*
UAP
UAP
UAP
UAP
LP4*
LP4
LP7
33
33
33
33
15
20
15
20
33
35
35 or 40
35 or 40
# cycles
unspliced
Cb splice
unspliced
ECO
unspliced
ECO
initial RACE
nested RACE
initial RACE
nested RACE
Cb splice
unspliced
Cb splice
Product
301
228
301
448
567
448
>379
>342
>591
>236
228
567
368
486
1087
448
Size
Suppl 3e
6b, 6c
6b, 6c
2d
2d
Suppl 3e
Suppl 3d
3c, 3d, 4c
3a, 3b, 4b
Figure
All PCRs had an initial denaturation step (94C x 3 min); followed by # cycles of 94C x 30 sec
denaturation, 57C x 45 sec annealing, and 72C x 60-70 sec extension; with a final extension
step (72C x 7 min)
Notes:
LP14
LP15
LP11
LP12
3RACE pA6
Triplex RT-PCR
LP5
LP10
3RACE pA1
LP8
LP14
LP15
RT-PCR
Triplex RT-PCR
LP13
RT-PCR
LP8
LP13
LP6
RT-PCR, gPCR
Triplex RT-PCR
LP8
RT-PCR, gPCR
LP4
Primers
FOR
REV
Experiment
78
5 sequence
...
...
...
...
...
ACTGACTGCAC|GTGAGTCGCTCCGTC
CCCTCCGGCGGGAGCT|CGCGGCGCGC
TCGCGGCGCGCCCCCGT|GCGCGGGCG
TCGCGGCGCGCCCCCG|TGCGCGGGCG
GGCCCTCCGGCGGGAGCTCGC|GGCGC
TM
[639]
[71]
[85]
[134]
[122]
del
TGTTTTGTAG|GTTTCATGAGTGGACATC
|CGCGGCGCGCAGGCGTCTGGCGGCCAAC
|GCGCGAGCGGCGCCGCATGCAGGGGCTG
|TGCGCAGGGTGGTGCCGCAGTGGGGCCA
GG|GGCTGAACACGGCGTTCGACCGGCTG
3 sequence
... AAGTCGGCCTGCAAACCCCAC|GGCCC
... ATGAAGTCGGCCTGCAA|ACCCCACG
...
...
...
...
...
...
...
...
...
...
CGTGGATGAAGTCGGCCTG|CAAACC
GAAAGGCTTTCTAT|CCCCGACCCCC
CTCCGGCGGGAGCTC|GCGGCGCGCC
CTCCGGCGGGAGCTC|GCGGCGCGCC
CTCCGGCGGGAGCTC|GCGGCGCGCC
CGCGCCCCCGTGCGCGG|GCGCAGCC
CTCGCGGCGCGCCCCCG|TGCGCGGGC
CCCCACGGCCCTCCGGCGG|GAGCTCG
CCCTCCGGCGGGAGCT|CGCGGCGCGC
TGCAAACCCCACGGCCCT|CCGGCGGG
[746]
[605]
[106]
[106]
[70]
[85]
[103]
[110]
[71]
[155]
[70]
[235]
|TAATCCTAGCGTCATTCAGGAGGTGGA
|CCCCTACCTCCCTTTCCCGGGTGCTAG
|GCGGCGCCGCATGCAGGGGCTGAACAC
|GCGGCGCCGCATGCAGGGGCTGAACAC
|CGCGGCGCGCAGGCGTCTGGCGGCCAA
GCGCGA|GCGGCGCCGCATGCAGGGGCT
|TGCAGGGGCTGAACACGGCGTTCGACC
GCGC|GAGCGGCGCCGCATGCAGGGGCT
|CGCGGCGCGCAGGCGTCTGGCGGCCAA
|CCGGCTGCGCAGGGTGGTGCCGCAGTG
...
...
...
...
...
...
...
...
...
...
|GGCCCGGGCGGCTGGAGAGCGCGGCGCG ...
CTGCAG|ATGGCGCTCAGCTACATCATCG ...
...
...
...
...
...
Spurious RT-PCR products are numbered to match Figure 5. The highlighted text shows
areas of micro-homology, which can promote RT template switching. The size of the
deletion [bp] and breakpoints (|) are indicated.
3
4
5
6
7
8
9
10
11
12
1
2*
ECO
InC
E12P20
32
9
No.
CHAPTER III
Abstract
The basic helix-loop-helix (bHLH) transcription factor Math5 (Atoh7) is
transiently expressed during early retinal histogenesis and is necessary for
retinal ganglion cell (RGC) development. Using nucleoside pulse-chase
experiments and clonal analysis, we determined that progenitor cells activate
Math5 during or after the terminal division, with progressively later onset as
histogenesis proceeds. We have traced the lineage of Math5+ cells using mouse
BAC transgenes that express Cre recombinase under strict regulatory control.
Quantitative analysis showed that Math5+ progenitors express equivalent levels
of Math5 and contribute to every major cell type in the adult retina, but are
heavily skewed toward early fates. The Math5>Cre transgene labels 3% of cells
in adult retina, including 55% of RGCs. Only 11% of Math5+ progenitors develop
into RGCs; the majority become photoreceptors. The fate bias of the Math5
cohort, inferred from the ratio of cone and rod births, changes over time, in
parallel with the remaining neurogenic population. Comparable results were
obtained using Math5 mutant mice, except that ganglion cells were essentially
79
absent, and late fates were overrepresented within the lineage. We identified
Math5-independent RGC precursors in the earliest-born (embryonic day 11)
retinal cohort, but these precursors require Math5-expressing cells for
differentiation. Math5 thus acts permissively to establish RGC competence
within a subset of progenitors, but is not sufficient for fate specification. It does
not autonomously promote or suppress the determination of non-RGC fates.
These data are consistent with progressive and temporal restriction models for
retinal neurogenesis, in which environmental factors influence the final histotypic
choice.
Introduction
The seven major cell types in the vertebrate retina (rod and cone
photoreceptors; amacrine, horizontal and bipolar interneurons; Mller glia; and
ganglion cells) develop from a common pool of progenitors (Turner and Cepko,
1987; Turner et al., 1990) that are established when the optic vesicles invaginate
to form bilayered optic cups (Goldowitz et al., 1996). The inner layer of each
optic cup consists of proliferative retinal progenitor cells (RPCs), which are
arranged as a pseudostratified epithelium. These RPCs begin to permanently
exit mitosis and differentiate around embryonic day 11 (E11) in the mouse.
Retinal neurons and glia are fully formed by postnatal day 21 (P21) and are
arranged in a highly ordered tri-laminated structure (Rodieck, 1998). The outer
nuclear layer (ONL) consists of photoreceptors while the inner nuclear (INL) and
ganglion cell (GCL) layers are populated by interneurons, glia and ganglion cells.
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The mechanism of cell fate determination how these diverse cell types are
generated from an initially homogeneous progenitor population remains poorly
understood.
Birthdating experiments, in which [3H]-thymidine was used to mark the
terminal S phase of progenitor cells, have established a characteristic order for
the emergence of different retinal cell types during histogenesis (Carter-Dawson
and LaVail, 1979; Rapaport et al., 2004; Sidman, 1961; Young, 1985a). In all
vertebrate species examined, retinal ganglion cells are the first-born neurons
(Altshuler et al., 1991). In mammals, these are followed by horizontal cells,
cones, amacrines, rods, bipolar cells and Mller glia, in descending birth order.
There is considerable overlap in the distribution of birthdates among cell types,
particularly for rod photoreceptors, which are born over an extended period (E13P7 in mice) and are most abundant. Moreover, as a subclass, displaced
amacrines, located in the mammalian GCL, are born earlier than amacrines in
the INL (LaVail et al., 1991; Reese and Colello, 1992).
Lineage tracing experiments in rodents and frogs show that individual
retinal progenitors are multipotent, giving rise to clones with heterogeneous cell
type composition and size, and that the histogenic potential of the progenitor pool
is gradually restricted over time (Holt et al., 1988; Turner and Cepko, 1987;
Turner et al., 1990; Wetts and Fraser, 1988; Wong and Rapaport, 2009). The
absence of a strict hierarchical relationship among cell types suggests that fate
determination in the retina is a stochastic process (Gomes et al., 2011; Livesey
and Cepko, 2001). The observation of discordant two-cell clones in rodent
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lineage marking studies indicates that at least some cell fate decisions occur
during or after the terminal division, and may be subject to environmental
influence (Turner and Cepko, 1987). Indeed, multiple extrinsic factors have been
shown to alter the ratio of retinal cell types generated from progenitor pools
(Altshuler et al., 1991; Ezzeddine et al., 1997; Fuhrmann et al., 1995; Yang,
2004; Young and Cepko, 2004).
Heterochronic mixing experiments, in which early and late retinal cells are
co-cultured in unequal ratios, have shown that progenitors have a limited
capacity to shift their fate forward or backward in sequence, and suggest that
competence is fundamentally a cell-intrinsic property (Belliveau and Cepko,
1999; Rapaport et al., 2001; Reh, 1992; Watanabe and Raff, 1990). Likewise,
single-cell dissociation studies have shown that the fates of retinal progenitors,
including post-mitotic cells, change over time and are intrinsically programmed
(Adler and Hatlee, 1989; Cayouette et al., 2003; Reh and Kljavin, 1989). Thus, it
is likely that cell-intrinsic factors, expressed by progenitors in a prescribed
temporal order, work in concert with extrinsic factors in the retinal
microenvironment to guide cell fate decisions and ensure proper ratios of each
cell type.
The basic helix-loop-helix (bHLH) transcription factor Math5 (Atoh7) was
identified on the basis of its homology to Drosophila Atonal (Brown et al., 1998),
which plays a critical role in the specification of R8 photoreceptors in the eye
imaginal disc (Frankfort and Mardon, 2002; Hsiung and Moses, 2002; Jarman,
2000; Sun et al., 2003). The mouse Math5 gene contains a single exon (Prasov
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sequences of the solitary Math5 exon (AF418923) and cloned into the SalI and
XhoI sites of pGSU-Cre (Cushman et al., 2000). The resulting A-Cre-B
cassette was inserted into the XhoI site of shuttle plasmid pLD53GFP10 as a
2.4 kb SacI-XhoI fragment and verified by DNA sequencing. Shuttle plasmid
pLD53GFP10 was derived from pLD53.SC1 by partial SpeI digestion and
insertion of a XhoI linker in place of the 3.5 kb EGFP fragment. We then targeted
RP23-328P3 with the Math5>Cre shuttle vector pLD53ACreB to obtain
ampicillin- and chloramphenicol-resistant cointegrates (Gong et al., 2002).
These were resolved by selection on TYE (tryptone-yeast extract) agarose plates
with chloramphenicol and 10% (w/v) sucrose. Two recombinant Math5>Cre BAC
clones were recovered and verified by PCR and pulsed-field gel electrophoresis
(PFGE) Southern analysis.
Purified circular DNA from Math5>Cre BAC clone RP23-328P3-D1-68 was
injected into fertilized (SJL/2 C57BL/6J) F2 oocytes by the UM Transgenic
Animal Core Facility. Nine transgenic founders were identified by Cre-specific
and BAC vector-insert junctional PCRs. Transgene copy number was
determined by Southern analysis, using an upstream Math5 genomic probe that
hybridizes equally well to 3.5 kb BAC and 6.5 kb mouse chromosomal EcoRI
fragments. Transgene integrity was evaluated by Southern analysis following
NotI digestion and PFGE. Transgenic offspring were genotyped using PCR
primers within the Cre-pA cassette.
Math5>Cre mice (line 872 or 360) were crossed to Z/AP (JAX stock
003919, (Lobe et al., 1999) and R26floxGFP (JAX stock 004077, (Mao et al.,
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88
89
90
Math5>Cre; R26floxGFP adult eyes. For P1 counts, the fraction of lineagelabeled RGCs was determined in retinal flatmounts from 3 eyes. The fraction of
each cell type descending from Math5+ progenitors, and the fraction of Math5+
progenitors giving rise to each cell type, were calculated based on detailed retinal
cell counts reported for adult C57BL/6J mice (Jeon et al., 1998). For lineage
tracing in the absence of Math5 gene function, labeled cells were counted in 23
fields (200X magnification) representing 6 adult eyes.
Dual reporter concordance
To assess Math5>Cre efficiency and heterogeneity among Math5+
progenitors, we crossed Math5>Cre; Z/AP mice to homozygous R26floxGFP
mice. Retinal sections from 3-4 week-old triple transgenic offspring (Math5>Cre;
Z/AP; R26floxGFP) were immunostained for GFP and hPLAP. Single- and
double-labeled cones, rods, amacrines and GCL neurons were counted in 18
fields (200X magnification) representing 4 eyes. To calculate concordance, we
divided the number of double-labeled cells by the total number of labeled cells.
Concordance was evaluated statistically using Cohens test (Cohen, 1960).
Birthdating and window labeling studies
To identify Math5 descendants exiting mitosis before P0, we performed a
cumulative BrdU labeling experiment (Miller and Nowakowski, 1988). Pregnant
dams carrying Math5>Cre; Z/AP embryos were given a single BrdU injection
(100 g/g body mass) on day E10.5 and provided with drinking water containing
500 g/mL BrdU and 1% sucrose (pH 7.0) until birth (Mayer et al., 2000). To
92
maximize labeling efficiency, water bottles were protected from light and replaced
daily. Retinal sections from 3-week-old offspring were immunostained for BrdU
and hPLAP.
To monitor how the fates of Math5+ progenitors exiting mitosis change
during development, we performed birthdating (pulse-labeling) experiments.
Pregnant dams carrying Math5>Cre; Z/AP embryos were given a single BrdU
injection (as above) on day E14.5, E15.5, E16.5 or E17.5 of development. Eyes
from 3-4 week-old mice were stained with BrdU and hPLAP antibodies, and PNA
lectin. The total number of cones (PNA+) and the number of hPLAP+ and/or
BrdU+ photoreceptors were counted in 14 central retinal fields (200X
magnification), corresponding to 3 eyes for each time-point. For birthdating
lineage-marked photoreceptors in the absence of Math5 function, we followed
the same protocol as above. We immunostained Math5>Cre; R26floxGFP;
Math5 -/- retinas for BrdU and GFP, and counted 7 fields (200X magnification)
from 2-4 eyes for each time-point. The fraction of lineage-marked and birthdated
cones was calculated directly from cell counts. The fraction of labeled rods was
estimated using a 35.2 rod-to-cone ratio for wild-type mice, based on C57BL/6
data (Jeon et al., 1998), and a 12.1 ratio for Math5 mutants (SEM = 0.8 based on
n = 5 animals, 71 fields at 200X magnification).
To determine the contribution of Math5+ cells to the early-born (EB) cohort
of neurons, we performed pulse- and window-labeling experiments at the onset
of neurogenesis. For pulse-labeling, gravid dams carrying Math5>Cre;
R26floxGFP embryos were given a single injection of EdU at day E11, and eyes
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from the resulting pups were harvested at P1. Whole retinas were stained for
GFP and EdU, flatmounted, and imaged as confocal Z-stacks through the
ganglion cell layer. The fraction of early-born cells in the Math5 lineage (EdU+
GFP+ / EdU+) was determined from 4 eyes representing 4 mice.
For window labeling (Repka and Adler, 1992), pregnant dams carrying
Math5 +/- and -/- embryos were given EdU on day E11, as a single injection or
two injections 12 hrs apart. No difference was apparent in the extent of EdU
labeling between these schedules. Dams were then given a single injection of
BrdU on E12 and provided with BrdU in the drinking water until harvest at E12.5.
Early-born cells (EdU+ BrdU-) were counted from 3-4 embryos of each genotype,
representing 1-3 litters, and scored for gal or Brn3b immunoreactivity.
Statistical error is reported as the binomial standard deviation. Labeled fractions
were compared using Fishers exact test (Fisher, 1925).
Retinal explants and clonal analysis
Retinal explant culture and retroviral infections were performed using
established methods, which favor RGC survival (Hatakeyama and Kageyama,
2002; Wang et al., 2002b). Math5 lacZ/+ retinas were dissected from E12.5 or
E13.5 eyes, removing sclerae, pigmented epithelium (RPE) and lens tissue, and
were flattened onto 5 mm Nucleopore polycarbonate membranes (0.4 m pore
size, GE Healthcare, Piscataway, NJ). These explants were placed on Transwell
inserts (Corning) in 2-cm dishes containing neurobasal media (Invitrogen) with
1X B27 and N2 supplements, glutamine (0.4 mM), BDNF (50 ng/mL, Peprotech,
Rocky Hill, NJ), CNTF (10 ng/mL, Peprotech), penicillin (50 U/mL), streptomycin
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(50 g/mL), and gentamicin (0.5 g/mL), and cultured at the gas-media interface
at 37C and 5% CO2.
MIG retroviral stocks (Van Parijs et al., 1999) were generated by
transfecting MSCV-IRES-GFP plasmid DNA into the Phoenix ecotropic
packaging cell line (Pear, 2001; Swift et al., 2001) and titered on NIH3T3
fibroblasts. Filtered viral preparations (~8x105 colony-forming units/mL)
containing polybrene (hexadimethrine bromide, 0.8 g/mL, Sigma Aldrich, St.
Louis, MO) were added directly to the explant surface in one drop (25 L) to
infect mitotic cells. After 2 days in vitro (DIV), half of the media was replaced
with fresh media. After 3 DIV, explants were fixed for 30 min in 4% PFA,
cryoprotected in 30% sucrose, and frozen in OCT. Thick (30 m) sections were
immunostained for gal and GFP. For each time-point, the size and composition
of clones was determined by 3-dimensional analysis of confocal Z-stacks.
Clones were defined as clusters of GFP+ cells directly apposed to each other
(within 2-3 m) and separated by at least 4 cell bodies from any other GFP+
cells. Only clones containing at least two GFP+ cells and one gal+ cell were
scored. Previous studies have shown that the average progenitor cell cycle
length is 14-16 hrs at this stage (Alexiades and Cepko, 1996; Sinitsina, 1971),
permitting 4-5 divisions during the 72 hr culture period. Accordingly, the largest
clones in each set of explants contained 8-16 cells, reflecting a minimum of 3-4
divisions in vitro.
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Results
Math5 is transiently expressed by early retinal progenitors during or after
their terminal cell cycle
As a first step to determine the mechanism of Math5 action, we defined
the timing of Math5 expression during retinal development by quantitative PCR
(Fig. III-1A). Math5 mRNA increases rapidly at E11, peaks between E12.5 and
E14.5, and declines gradually after E14.5. This temporal profile is consistent with
RNA in situ hybridization data (Brown et al., 1998) and closely resembles
birthdating curves for RGCs (Drager, 1985; Young, 1985a). These data suggest
Math5 acts transiently during early retinal neurogenesis.
The cellular distribution of Math5 mRNA and Math5-lacZ activity across
the retinal epithelium (Brown et al., 2001) is consistent with Math5 transcription in
actively proliferating and/or postmitotic cells. Both patterns have been reported
for different bHLH genes during neurogenesis (Kageyama and Nakanishi, 1997).
Indeed, the closely related gene Math1 is expressed in mitotic cells in the
developing cerebellum (Helms et al., 2000) and in postmitotic cells in the inner
ear (Chen et al., 2002). In frog, zebrafish and chick retinas, orthologous Ath5
genes are expressed in progenitors during their last cell division (MatterSadzinski et al., 2001; Perron et al., 1998; Poggi et al., 2005).
To determine the onset of Math5 expression in individual mouse retinal
progenitors, we immunostained E13.5, E15.5, and E16.0 eyes from Math5 +/(lacZ/+) and/or Math5 -/- (lacZ/lacZ) embryos for -galactosidase (gal), the cell
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RGCs (Brown et al., 2001). In double transgenic Math5>Cre; Z/AP embryos, the
alkaline phosphatase (hPLAP) marker first appeared at E12.5 in differentiating
RGCs and the developing optic nerve (Fig. III-2D), whereas no hPLAP was
detected in control embryos carrying Z/AP alone (Fig. III-2C). At later
developmental stages, some other cell types were labeled with hPLAP (e.g.
photoreceptors at P0.5 in Fig. III-2D, arrowhead). As expected, hPLAP was only
detected in the adult retina and brain, in known Math5 RNA expression domains.
In the central nervous system, the hPLAP reporter marks neurons in the auditory
hindbrain and cerebellum (Saul et al., 2008) and reveals all known RGC
projections (Rodieck, 1998; Simpson, 1984), including those extending to the
superior colliculi, lateral geniculate bodies, suprachiasmatic nuclei, and the
accessory optic tracts (Fig. III-2E).
In the E15.5 retina, a comparison of the spatial and temporal patterns for
Math5 mRNA, Math5-lacZ and hPLAP (Fig. III-2F) is consistent with a direct role
for Math5 in RGC development and highlights the inherent time delay associated
with Cre protein synthesis, excisional activation of the Z/AP reporter, and
expression of the hPLAP enzyme (Nagy, 2000). Considering the dynamics of
retinal interkinetic nuclear migration (Baye and Link, 2008), these results suggest
there is a burst of Math5 expression in progenitors exiting the cell cycle. If Math5
is exclusively made during the last division, lineage-marked cells should never
re-enter S phase. To test this prediction, we analyzed E13.5 Math5>Cre;
R26floxGFP and E15.5 Math5>Cre; Z/AP embryos exposed to EdU or BrdU for
1 hr (Fig. III-2G,H). In E13.5 embryos after a 30 min chase, some Cre+ EdU+
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cells were present (33 of 394 Cre+ cells = 8.4 0.4% SD for n = 3 sections) and
these were restricted to the fresh neurogenic subset (33 of 223 Cre+ GFP cells
= 14.8 1.4% SD). No GFP+ EdU+ cells were observed in the same sections (0
of 309 GFP+ cells, upper limit 95% CI = 0.9%), due to the delay in the Cre-lox
system (Fig. III-2G). Likewise, in E15.5 embryos, there was no overlap between
hPLAP activity and any cell cycle marker (Fig. III-2H). Together, these results
strongly suggest that Math5 is expressed transiently during or shortly after the
terminal cell division. Math5 lineage cells do not re-enter the cell cycle.
Quantitative Math5 lineage analysis
To reveal the fates of Math5+ progenitors, we crossed Math5>Cre mice to
Z/AP and R26floxGFP reporter strains and examined mature retinas of 3-4 week
old offspring. We observed hPLAP+ cells distributed evenly across the central
and peripheral retinas of Math5>Cre; Z/AP mice, but staining was absent in
littermates carrying the Z/AP transgene alone (Fig. III-3A,B). Because hPLAP
protein is membrane-tethered, we could identify most retinal cell types by
morphology and laminar position. As expected, RGCs were abundantly labeled.
However, we also observed significant staining among rods, cones, horizontal
and amacrine cells (Fig. III-3A,C,D). The inner plexiform layer (IPL) was
intensely labeled due to hPLAP localization in RGC and amacrine dendrites. A
thorough survey revealed rare hPLAP+ Mller glia and bipolar cells (Fig. III-3C).
Importantly, no labeling was observed in retinal cell types that have a separate
developmental origin, such as vascular endothelial cells, pericytes, microglia and
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astrocytes, or in any other parts of the eye, including the anterior chamber and
RPE.
To systematically measure the fraction of lineage-marked retinal cells in
each class, we co-stained sections for hPLAP or GFP reporters and cell
type-specific markers. Equivalent results were obtained using Z/AP and
R26floxGFP reporters (see below) and different Math5>Cre lines (data not
shown). However, the intensity of expression varied among cell types. Z/AP is
strongly expressed in photoreceptors via the CAG promoter, whereas
R26floxGFP is weakly expressed by photoreceptors but strongly expressed by
other cell types. Consequently, we used Z/AP to count hPLAP+ and hPLAPcones (arrows in Fig. III-3D), and hPLAP+ rods (arrowheads in Fig. III-3D) in the
outer nuclear layer (ONL), and PNA lectin to distinguish cones from rods (Blanks
and Johnson, 1983). We then counted hPLAP+ bipolar cells and Mller glia (Fig.
III-3C) in the inner nuclear layer (INL) of the same sections. The labeled fraction
was calculated for each cell type using reference data for cell populations in the
adult mouse retina (Jeon et al., 1998). This fraction ranged from 31% for cones
to 1% for rods, and <0.1% for bipolar cells and Mller glia (Table III-1).
To evaluate horizontal, ganglion and amacrine cell types, we used the
R26floxGFP reporter, which co-localizes with cell type-specific markers in the
perinuclear cytoplasm. We identified horizontal cells by calbindin
immunostaining (Peichl and Gonzalez-Soriano, 1994) and their position at the
outer edge of the INL (Fig. III-3E). Twenty-nine percent of horizontals were
GFP+ (Table III-1). This value is significantly lower than that reported by Yang et
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al. (2003), but consistent with horizontal cell labeling data of Feng et al. (2010, cf.
Suppl. Fig. 3E) obtained using a Math5-Cre knock-in allele. RGCs were
distinguished from displaced amacrine cells (Hayden et al., 1980; Perry and
Walker, 1980) by retrograde rhodamine-dextran tracing of optic nerve axons.
Forty-three percent of neurons in the ganglion cell layer (GCL) were labeled with
rhodamine in this experiment (arrows, Fig. III-3F), in close accord with previous
data (Jeon et al., 1998). All other cells in the GCL were scored as displaced
amacrines (arrowheads, Fig. III-3F). The frequency of GFP labeling in the adult
retina was 55% for ganglion cells, 28% for displaced amacrines, and 9% for INL
amacrines (Table III-1). To evaluate the Math5 lineage fraction prior to the
normal period of RGC culling (Galli-Resta and Ensini, 1996), we performed a
similar analysis in early postnatal retinas, limited to the GCL (Fig. III-3G). The
fraction of GFP+ ganglion cells in P1 retinas (53 1%, n = 3, 948/1777 cells) was
similar to that observed in the adult (55 2%, Table III-1, Fishers exact test P =
0.3).
A clear pattern emerges from these data. First, Math5+ progenitors have
the potential to develop into all seven major retinal cell types. Second, the
distribution of Math5+ descendents differs from the retina as a whole (Fig. III-3I,
2 test with df = 7, P < 0.0001). Third, the labeling fraction of each cell type
(Table III-1) decreases according to the birth order (Carter-Dawson and LaVail,
1979; Rapaport et al., 2004; Sidman, 1961; Young, 1985a). Early-born cell types
RGCs, cones, horizontal cells and displaced amacrines frequently descend
from Math5+ progenitors, whereas late-born bipolar and Mller glial cells rarely
102
derive from Math5+ progenitors. INL amacrines are born during the middle
phase of retinal development, prior to the peak of rod births, and these cell types
have an intermediate labeling fraction. We estimate that 3% of adult retinal cells
descend from Math5+ progenitors (Table III-1). Fourth, only one in nine Math5+
descendants is a ganglion cell (11%). Because RGCs represent ~0.5% of
neuroretinal cells in adult mice (Jeon et al., 1998) and Math5 status does not
affect RGC survival between P1 and adulthood, Math5 descendants are 50-fold
more likely on average to develop as RGCs than the remaining neuroretinal
population (approx. 1 in 500). Fifth, 45% of ganglion cells are not marked by the
Math5>Cre transgene, suggesting the possibility of a substantial
Math5-independent RGC subpopulation. Although the fraction of GCL neurons
labeled by Math5>Cre (40%, Table III-I) approximates the RGC fraction (43%),
this value includes both RGC (24%) and displaced amacrine cell types (16%).
The fate of Math5 mutant cells
In mutant mice, the Math5 transcription unit is active, expressing lacZ
mRNA, but lineage-marked progenitors are blocked from developing as RGCs.
To determine the fates of these cells, we examined retinas from adult Math5 -/mice carrying Z/AP and Math5>Cre transgenes (Fig. III-3H). The extent of
hPLAP labeling in the mutant retina was roughly similar to wild-type (Fig. III-3A).
However, the fate profile within the Math5 lineage was different (2 test with df =
7, P < 0.0001). First, RGCs were absent, as expected, decreasing the amount of
IPL staining. Second, there was an obvious increase in late-born cell types
among the hPLAP+ neurons (Table III-S1). For example, rod photoreceptors
103
increase from 32% to 40% of the Math5 lineage. Labeled bipolar cells and Mller
glia were visible in most low power fields (200X magnification) of mutant mice,
but were difficult to find in wild-type Math5>Cre; Z/AP retinas (Table III-1),
consistent with results observed by Feng et al. (2010). This effect is more
striking if one considers that the total number of rods, bipolar cells and Mller glia
are decreased in Math5 mutants (Brown et al., 2001; Brzezinski et al., 2005). In
Math5 mutant mice, the cohort of progenitors expressing Math5>Cre does not
differentiate into RGCs, but retains competence to develop into any of the
remaining principal cell types.
Math5+ progenitors have equivalent Cre activity
Only a small fraction (11%) of the Math5 lineage develops into RGCs. In
principle, the Math5+ population may be heterogeneous, such that one group of
progenitors expresses high levels of Math5>Cre and develops as RGCs, while a
second group expresses low levels of Math5>Cre and adopts other fates (Fig. III4A). In this model, the low-level multi-lineage Math5>Cre expression could
represent priming (Hu et al., 1997) of the Math5 gene, or leaky transgene
expression, an over-reporting artifact that is not biologically meaningful
(Dymecki et al., 2002). Alternatively, all Math5+ progenitors may express
equivalent levels of Math5>Cre (Fig. III-4B), consistent with a permissive role for
Math5 in retinal development.
To test these alternatives, we examined retinas from triple transgenic
(Math5>Cre; Z/AP; R26floxGFP) adult mice, using the concordance of hPLAP
and GFP labeling in Math5 descendants as an indirect measure of Cre activity
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P0 should be weakly labeled. We found that essentially all hPLAP+ cells in the
central retina were heavily labeled with BrdU (98.8%), including rods
(arrowheads in Fig. III-5A), cones (arrows in Fig. III-5A), and INL and GCL
neurons (Table III-S3). Therefore, Math5+ rods, bipolars and Mller glia are born
at the leading edge of birthdating curves for these late cell types.
The fate profile of neurogenic cells emerging from the RPC population is
known to change over time, in response to intrinsic factors and environmental
signals (Livesey and Cepko, 2001; Rapaport et al., 2004; Young, 1985a). This
can occur through alterations in the fate bias of individual cells or the composition
of the RPC pool (heterogeneity). In principle, the Math5+ cohort may behave
similarly. The fate profile of these cells may be intrinsically programmed, or it
may vary depending on the time that an individual RPC exits mitosis and initiates
Math5 transcription. To test these alternatives, we compared the fates of Math5+
progenitors born on different days. Math5>Cre; Z/AP embryos were exposed to a
pulse of BrdU on E14.5, E15.5, E16.5 or E17.5 and their adult retinas were
examined by hPLAP, PNA, and BrdU staining (Fig. III-5B). A variety of
lineage-marked cell types were born on each of these days, including RGCs,
rods, cones, amacrines and horizontal cells, as well as rare late cell types
(arrowhead in Fig. III-5B). For quantitative analysis, we focused on
photoreceptors, which are relatively numerous and could be directly compared
within the ONL. At each time-point, we determined the fraction of hPLAP+ and
heavily BrdU+ rods and cones in the central retina (arrows in Fig. III-5B, Table IIIS4). The fraction of photoreceptors (rods plus cones) that were derived from
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Math5+ progenitors decreased between E14.5 and E17.5, from 20.6% to 4.3%
(Fig. III-S2, Table III-S4), in parallel with the decrease in the total number of
Math5+ cells.
The fate of the Math5+ cell population also changed significantly between
E14.5 and E17.5, together with the retina as a whole. Math5+ cells born on
E14.5 were >2 times as likely to develop into cones as compared to rods (136 vs.
64), whereas those born on E17.5 were >60 times as likely to develop into rods
as compared to cones (122 vs. 2, Table III-S4). The fates of progenitors inside
and outside the Math5 lineage shifted in parallel, as shown by plots of the coneto-rod ratio (Fig. III-5C), derived from birthdating curves (Fig. III-S2). This shift is
primarily determined by the overall decrease in cone births during this interval.
Math5+ cells appear to follow the same fate trajectory as other progenitors.
However, the ratio curves are displaced by one-half day. In comparison to other
neurogenic cells (hPLAP-) exiting mitosis on the same day in the same retinal
environment, Math5+ progenitors (hPLAP+) were three times more likely to
develop into cones. Surprisingly, similar results were obtained in the absence of
Math5 function, in mutant embryos carrying R26floxGFP and Math5>Cre
transgenes (Fig. III-5D).
These findings support three conclusions. First, the fate profile of Math5+
cells changes over time, similar to that of other retinal progenitors. Second, the
fate bias of Math5+ cells extends beyond RGC specification, influencing the
choice among alternative fates (e.g. cone vs. rod). Third, the bias among
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108
expected from the deficiency of RGCs in these mice, and confirms that the loss
of RGCs is an early event. We next determined the fraction of EdU+ BrdU- cells
that expressed Math5, using the lacZ allele (gal) as a short-term lineage tracer
(Wang et al., 1999). Surprisingly, only 20% of EdU+ BrdU cells were gal+, in
both Math5 +/- and Math5 -/- mice (Fig. III-6C). To independently test this result,
we exposed Math5>Cre; R26floxGFP embryos to a single pulse of EdU at E11,
harvested their retinas at P1, and determined that 28% of strongly EdU+ cells in
the GCL were GFP+ (Fig. III-6D,E). As a third test, we evaluated retinas from
early Math5-lacZ/+ embryos for coexpression of lacZ and Brn3b. The fraction of
gal+ RGCs was relatively low at E11.5, consistent with the EB analysis, but
increased from 20% to 60% between E11.5 and E13.5 (Fig. III-7). Taken
together, the results from these three experiments suggest that Math5 is
expressed by a subset of early neurogenic cells, and that only a fraction of
Brn3b+ RGCs generated at E11-13 derive from the Math5+ cohort.
The Math5-independent early-born cells may express other proneural
bHLH transcription factors in the Atonal family, such as Neurod1 or Neurog2. At
E11.5, Neurod1 was detected in a pattern that partially overlaps Math5-lacZ (Fig.
III-S3), consistent with mRNA in situ hybridization data (Hufnagel et al., 2010). A
similar overlap has been noted later in development (Kiyama et al., 2011; Le et
al., 2006). This may explain the small number of early-born Brn3b+ RGCs
present in Math5 -/- mice (Fig. III-6B), as Neurod1 can partially substitute for
Math5 function in RGC fate specification (Mao et al., 2008b).
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110
lineages, given their small size and time in culture. Indeed, all cells in these
small clones were postmitotic, as judged by expression of the cell cycle inhibitor
p27Kip1 (Dyer and Cepko, 2001a) (data not shown). Among 23 clones scored,
we observed both symmetric (Ngal-Ngal) (Fig. III-8B,C) and asymmetric (N-Ngal,
or possibly P-Ngal) (Fig. III-8D) patterns of Math5 expression. Of 23 informative
neurogenic divisions, 13 (57%) were symmetric with respect to Math5 expression
and 10 were asymmetric (Fig. III-8E). The fraction of symmetric divisions did not
differ significantly between the E12.5 and E13.5 explant time-points (0.64 vs.
0.50 respectively, Fishers exact test P = 0.7). Although few symmetric terminal
divisions are expected at this age in the retina as a whole, the high frequency
observed among the Math5+ cohort (Ngal-Ngal) confirms that early progenitors
are capable of N-N divisions in mice as in zebrafish (Poggi et al., 2005). Unlike
zebrafish, neurogenic divisions can be asymmetric with respect to Math5
expression in mice. These findings confirm that many retinal progenitors express
Math5 after terminal M phase.
Discussion
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112
113
114
The fraction of unmarked RGCs (~45%, Table III-1) is 10-fold greater than
the number of RGCs that survive in Math5 -/- mice (~4%)(Lin et al., 2004). Apart
from Cre inefficiency (noted above), there are two possible explanations for this
discrepancy. First, RGCs derived from Math5+ progenitors may have a survival
advantage during neonatal period (P0-P10) of ganglion cell apoptosis (Young,
1984). However, the deficiency of Math5-independent RGCs in Math5 mutants
was clearly evident early in retinal histogenesis, at E12.5 (Fig. III-6B,C), well
before the neonatal period of RGC culling. In addition, the fraction of Math5+
RGCs in P1 and adult retinas was the same, making this mechanism unlikely.
Second, Math5 lineage cells may have a substantial non-autonomous role in
RGC fate specification or early differentiation. These cells may represent
pioneering neurons (Pittman et al., 2008; Raper and Mason, 2010), which
promote axon pathfinding and fasciculation within the retina (Erskine and
Herrera, 2007; Oster et al., 2004) and survival of Math5-independent RGCs. In
the absence of Math5, cells in the inner retina undergo apoptosis during
midgestation and surviving RGCs have severe pathfinding defects (Feng et al.,
2010; Kiyama et al., 2011; Moshiri et al., 2008); Chapter V). Most likely, Math5+
progenitors may favor the formation or survival of other RGCs by para- or
juxtacrine signaling. Further work is needed to clarify molecular differences
between the Math5+ cohort and other cells in the early retina.
Math5 is made by progenitors exiting the cell cycle
We have determined the precise relationship between onset of Math5
expression and the cell cycle status of retinal progenitors (Fig. III-9A). At early
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stages (<E14), Math5-lacZ was detected in some G2/M phase progenitors but
was otherwise present only in non-proliferating cells. Based on the length of G2
phase (~ 2 hrs) (Sinitsina, 1971) and our analysis of retinal cell cycle kinetics in
E13.5 Math5>Cre; R26floxGFP embryos, following a 30 min EdU pulse (Fig. III2G), we conclude that at least 15% (and up to 60%) of newly Math5+ cells (Cre+
GFP-) initiate expression before terminal M phase. During later stages (>E15),
Math5 was exclusively expressed in post-mitotic cells. Math5 lineage cells did
not re-enter the cell cycle at any stage, regardless of the Math5 genotype. This
comprehensive analysis reconciles previous disparate observations regarding
the timing of Math5 expression (Brown et al., 1998; Le et al., 2006; Yang et al.,
2003), including RNA profiling of single retinal cells (Trimarchi et al., 2008). In
recent studies, an HA epitope-tagged Math5 allele was expressed with similar
kinetics in early E12.5-E14.5 embryos, but was detected in more S, G2, and M
phase cells than our Math5-lacZ allele (Feng et al., 2010; Kiyama et al., 2011;
Wu et al., 2012). This is comparable to zebrafish, where ath5-GFP expression
initiates during terminal S/G2 (Poggi et al., 2005), and is consistent with results
obtained in frog and chick (Kay et al., 2001; Matter-Sadzinski et al., 2001; Perron
et al., 1998; Poggi et al., 2005).
The variable timing of Math5 expression was supported by detailed clonal
analysis. We observed symmetric Math5-lacZ expression in 13 of 23 informative
divisions (56%, NMath5-NMath5) and asymmetric expression in the remaining clones
(P/N-NMath5). This frequency is convergent with cell cycle kinetic data discussed
above. Together, these findings suggest that early progenitors giving rise to
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have significantly fewer late-born cells, presumably due to loss of Shh. Because
all Math5 lineage cells retain early birthdates in these retinas (Fig. III-6C, Fig. IIIS2), this cohort appears expanded and skewed toward late cell fates. Taken
together, we conclude Math5 does not directly control the acquisition of multiple
retinal cell fates (Feng et al., 2010). Instead, Math5 has an active role in RGC
fate specification, as a competence (permissive) factor, and a passive or minor
role in the selection of alternative (non-RGC) fates.
Mechanisms of fate determination in the mouse retina
Retinal cell fate choice, differentiation and survival are jointly controlled by
intrinsic and extrinsic factors (Livesey and Cepko, 2001). As a nuclear bHLH
protein, Math5 is an intrinsic factor. It is necessary but not sufficient for RGC
development. During retinogenesis, nine-fold more Math5+ cells are produced
than develop into RGCs (Fig. III-3, Table III-1). These cells have a different fate
bias than other neurogenic cells in the same environment (Fig. III-5). This
property is conferred upstream of Math5. The development of RGCs from
Math5+ cells may require the presence of positive cofactors or the absence of
inhibitors. Soluble factors and cell-cell signaling are known to negatively regulate
RGC genesis, including factors secreted by nascent RGCs (Austin et al., 1995;
Belliveau and Cepko, 1999; Waid and McLoon, 1998; Zhang and Yang, 2001),
and these may act on Math5+ cells. Together, our data suggest that the Math5+
cohort is influenced by intrinsic and extrinsic factors.
Our finding that Math5+ progenitors born on the same day can give rise to
early or late cell types (Fig. III-5) is consistent with a progressive restriction
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model for retinal neurogenesis, in which the progenitor pool is initially multipotent,
but gradually loses competence to form early cell types (Pearson and Doe, 2003;
Shen et al., 2006). This model is favored by heterochronic co-culture
experiments (Reh, 1992; Watanabe and Raff, 1990) and Ascl1 (Mash1) lineage
analysis. Mouse Ascl1+ progenitors form all retinal cell types except RGCs
(Brzezinski et al., 2011) and may represent the first competence-restricted state.
However, our results are also consistent with a temporal restriction model, in
which progenitors proceed unidirectionally in time through a relatively fixed series
of competence states (Wong and Rapaport, 2009).
The reservoir of neurogenic cells that are competent to form RGCs greatly
exceeds the final number. Likewise, the period of RGC competence extends
beyond the normal time envelope for RGC births in rat and chick (James et al.,
2003; Silva et al., 2003). This excess capacity, which includes Math5+ and
Math5- cells, and the fate plasticity of Math5+ cells may serve to enhance the
robustness of RGC development and ensure an appropriate histotypic profile in
the mammalian retina.
Acknowledgements
The authors are grateful to Thom Saunders, Maggie van Keuren and the
UM transgenic animal model core for generating the BAC transgenic animals; to
Sue Tarl and Dellaney Rudolph for technical support; to Nadean Brown for in
situ hybridization data; to Nathaniel Heintz for BAC targeting plasmids and
strains; to Sally Camper for the nlsCre plasmid; and to Sean Morrison for the
MIG retroviral construct. The authors thank Chris Edwards, the UM microscopy
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and image analysis laboratory staff, Rafal Farjo and Mohammad Farah for
technical advice. The authors are grateful to Nadean Brown, Chris Chou, David
Turner, Matt Wilken, Julia Pollak, Anna La Torre, and Yumi Ueki for valuable
discussions and critical reading of the manuscript. This research was funded by
National Institutes of Health (NIH) R01 grant EY14259 (TG). JAB and LP were
supported by NIH T32 grants EY13934 (JAB, LP), GM07544 (JAB), and
GM07863 (LP).
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125
Figure III-3. Math5+ progenitors contribute differentially to all retinal cell types.
Math5>Cre mice were crossed to Z/AP (A-D) or R26floxGFP reporter (E-G)
strains. (A) In Math5>Cre; Z/AP mice, hPLAP+ descendants of Math5+
progenitors represent 3% of adult retinal cells (see Table III-1) and are present in
every cell layer. (B) Z/AP-only control retinas have no hPLAP activity. (C)
Math5+ descendants, detected by hPLAP immunostaining, include horizontal (h),
ganglion (rgc), displaced amacrine (da), INL amacrine (a), bipolar (b), rod (r),
cone (c) and Mller glial (m) cells. (D) Math5+ cone (arrows) and rod
(arrowheads) photoreceptors are distinguished by co-labeling with anti-hPLAP
and cone-specific PNA lectin. Non-specific labeling of pigment epithelium and
choroid reflects mouse IgG crossreactivity. (E-G) In Math5>Cre; R26floxGFP
mice, Math5+ horizontal cells (E, arrows) are marked by GFP and calbindin
immunoreactivity. The arrowhead shows a solitary Math5+ bipolar cell. (F-G)
Math5+ RGCs (arrows) and displaced amacrines (arrowheads) in the GCL are
shown in adult retinal sections (F) or P1 retinal flatmounts (G). RGCs are
distinguished by retrograde labeling of optic nerve axons with rhodamine dextran.
There is no difference in the GFP+ fraction of rhodamine dextran-labeled RGCs
between these two ages. (H) The fate of Math5>Cre- expressing progenitors in
Math5 -/- mice. hPLAP+ cells are distributed throughout the retina, but RGCs are
lacking. Vitreal hemorrhages (arrowhead) are common in Math5 -/- mice. (I) The
distribution of cell fates in the entire retina (from Jeon et al., 1998), in the Math5
lineage of wild-type mice, and in the Math5 lineage of knockout mice. The Math5
lineage is biased toward early-born cell types (RGC, horizontal, cone), although
rods are the most common fate adopted by Math5+ cells. In the Math5 knockout,
lineage-derived cells adopt all retinal fates except for RGCs. hPLAP, human
placental alkaline phosphatase; o, outer nuclear layer; i, inner nuclear layer; g,
ganglion cell layer. Scale bars, 100 m in A-B, H; 50 m in C-G.
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127
Figure III-4. All Math5>Cre progenitors express similar levels of Cre, regardless
of cell fate. Math5>Cre lineage analysis was performed using Z/AP and
R26floxGFP reporters simultaneously, to evaluate the heterogeneity of Math5
expression among progenitors. This analysis assumes that the probability of
reporter activation in a given cell is determined by the cumulative amount of Cre
recombinase expressed by that cell. (A-B) Two models for Math5 (Cre)
expression. (A) Bimodal expression. In this model, Math5+ progenitors giving
rise to non-ganglion cell types express Cre weakly (left peak), so reporter
activation in these cells is inefficient, and consequently few of their descendants
co-express GFP and hPLAP. RGCs in the same retinas express Cre strongly
(right peak) and are expected to have high concordance. (B) Uniform
expression. In this model, every Math5+ progenitor expresses Cre strongly, so
concordance is very high for all cell types (B, right). (C) Retinas of adult
Math5>Cre; Z/AP; R26floxGFP mice immunostained for hPLAP and GFP.
Double-labeled cells (arrows) greatly outnumber single-labeled cells
(arrowheads). (D) The observed concordance between hPLAP and GFP
reporters was ~80%, which is significantly greater than expected by chance
(Cohens > 0.7). This value was similar for all cell types, indicating that Math5
is expressed at uniform levels by a subpopulation of progenitor cells, only some
of which develop as RGCs. The labeled fractions () are based on data in Table
III-1. GCL includes RGCs and displaced amacrines; INL Am, inner nuclear layer
amacrine. Scale bar, 50 m.
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129
Figure III-5. The fate distribution of Math5+ progenitors changes over time. (A)
Cumulative BrdU labeling experiment. Math5>Cre; Z/AP embryos were
continuously exposed to BrdU from E10.5 to P0 and their retinas were collected
at P21. Nearly all Math5+ descendants (hPLAP+) are heavily labeled with BrdU,
indicating that the majority exited mitosis before P0, including lineage-labeled
cones (arrows) and rods (arrowheads). There is a distinct gradient of BrdU
labeling (birthdates) within the inner and outer nuclear layers, such that cells with
nuclei closest to the lens have earlier birthdates (brightest BrdU signal). (B)
Pulsed BrdU labeling experiment. Math5>Cre; ZAP embryos were transiently
exposed to BrdU at E15.5. Adult retinas were stained with hPLAP and BrdU
antibodies and PNA lectin. Math5+ cone (hPLAP+ PNA+ BrdU+, arrow) and
bipolar (hPLAP+ BrdU+, arrowhead) cells are indicated. (C) Cone-rod ratio plots
for birthdated hPLAP+ (red), hPLAP (blue) and combined (black) photoreceptor
groups. The ratio of cone-to-rod births decreases steadily between E14.5 and
E17.5 for hPLAP+ and hPLAP populations. The curves are parallel, indicating
that the fate of Math5+ cells changes over time, similar to other retinal
progenitors. However, the cone-to-rod ratio is 3-fold higher for Math5+
progenitors at every time point, suggesting that these cells have a fixed cone vs.
rod bias, or are shifted by 0.5 days, compared to other neurogenic cells
(hPLAP) in the same retinal environment. (D) Cone-rod ratio plot for birthdated
GFP+ (red), GFP (blue) and combined (black) photoreceptor groups in Math5 /-; Math5>Cre; R26floxGFP mice. Scale bar, 50 m.
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131
Figure III-6. Math5 marks many of the earliest born cells in the retina. (A-C)
Window labeling analysis. (A) Embryos were exposed to pulses of EdU at E11
(onset of neurogenesis) and E11.5, and to continuous BrdU from E12 to E12.5.
Progenitors (RPCs) that continue to cycle through E12.5 are EdU+ BrdU+, while
cells that have exited mitosis between E11 and E12 are EdU+ BrdU, and
represent the earliest born cohort of retinal neurons. (B-C) Sections through the
neural retina (brackets). (B) Most early-born cells in Math5 +/- mice adopt RGC
fate (EdU+ BrdU- Brn3b+, arrows). The Brn3b- cells in this cohort are likely to
include horizontal cell precursors (arrowheads). Few Brn3b+ RGCs (arrows) are
present in Math5 -/- embryos, and the abundance of non-RGC fates increases
accordingly (arrowheads). (C) Early-born Math5-lacZ (EdU+ BrdU- gal+,
arrows) and gal (arrowheads) cells are shown in Math5 +/- (top) and Math5 -/(bottom) mice. Only ~20% of the early-born cohort expresses the Math5
transcription unit (gal+), in both genotypes. (D-E) Birthdating analysis. (D) E11
Math5>Cre; R26floxGFP embryos were exposed to a single EdU injection and
analyzed at P1. (E) Flatmounted retinas were stained for EdU and GFP and
imaged through the GCL. Strongly EdU+ cells mark the earliest born retinal
neurons. Confocal projection image (6-10 m) shows EdU+ GFP+ (arrow) and
EdU+ GFP (arrowhead) cells. Only 28% of the GCL cells born at E11 are in the
Math5+ lineage. i.p., intraperitoneal; EB, early-born. Error bars represent the
binomial standard deviation. Scale bar, 50 m.
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Figure III-7. A subset of Brn3b+ RGCs derives from the Math5 lineage. (A-C)
Sections from embryonic Math5-lacZ/+ retinas co-stained for gal and Brn3b. At E11.5,
relatively few Brn3b+ cells are gal+ (A, arrow). The fraction of Brn3b+ cells expressing
Math5-lacZ (arrowheads) increases from E12.5 (B) to E13.5 (C). However, there
are many Math5-independent RGCs (arrows) at each age. (D) Histograms showing
the fraction of Math5+ cells among Brn3b+ RGCs and the fraction of Brn3b+ RGCs
within the Math5+ cohort. Error bars show the standard deviation (n = 3 sections).
The total number of cells counted at E11.5, E12.5 and E13.5 was 13, 228 and 667,
respectively. Scale bar, 50 m.
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135
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Figure III-9. Natural history of the Math5 lineage. (A) The timing of Math5
expression shifts during retinal histogenesis. RPCs (white) shift from a
proliferative (P-P) mode of division to stem (N-P) or terminal (N-N) modes, giving
rise to neurogenic cells (gray). These express Math5 (red) either during (S,
symmetric) or after (A, asymmetric) final mitosis. During early retinal
development (<E14), Math5 is frequently expressed during G2 phase of the last
cell cycle, generating two Math5+ daughters. During later stages (>E15), Math5
is exclusively expressed by post-mitotic cells. (B) The size of the neurogenic
(birthdated) population and proportion of Math5+ cells changes during
development. At the onset of neurogenesis (E11), Math5 is expressed by
20-30% of newborn cells. The number of Math5+ cells peaks during
midgestation (E14) and rapidly diminishes (E16), while the neurogenic population
as a whole continues to expand. The temporal profile for RGC birthdates follows
similar kinetics, and reflects Math5+ and Math5 populations. (C) The fate
spectrum of Math5 lineage (red) and other neurogenic (gray) cells in wild-type
and mutant mice. The thickness and shading of arrows denotes the relative
demographic contribution of these cohorts to the mature retina.
137
138
139
1,041
1,665
horizontal
amacrine
displaced
12
2,085
467
8,800*
12,900*
< 0.1
< 0.1
28
1,648#
173,000*
11
29
31
55
(a/b) x 100
(% of cell type)
13,920*
15,570*
3,592
4,914
1,265#
(b)
total
Math5 lineage
Aa100.0
2.9
7.4
a78.5
0.9
7.0
7.9
0.5
2.2
0.6
(c) x 100
(% of retina)
cell type
2.9 (f )
< 0.002
< 0.005
0.9
0.2
0.6
0.8
0.2
0.7
0.3
(% of retina)
Math5 lineage
100
< 0.1
< 0.2
32
20
29
23
11
(% of Math5 lineage)
cell type
Calculated from Jeon et al. (1998) and shown in Fig. 3I (left pie chart)
Among n = 8 eyes, the mean RGC labeling fraction SEM was 54 2%, with a range between 46 and 63%. The overall labeling fraction for the GCL was 40% (1167/2913 cells), which
represents 24% RGCs (700/2913 cells) and 16% displaced amacrines (467/2913 cells).
RGCs (identified by retrograde axon labeling) represent 43% of GCL neurons (1265/2913 cells). The remaining GCL cells were scored as displaced amacrines.
* Estimated using cell type ratios reported by Jeon et al. (1998) for adult C57BL/6J mice. The total number of cones counted in surveyed fields was multiplied by 35.2 to give the number
of rods, and by 3.32 for bipolar cells and 1.3 for Mller glia. The total number of GCL neurons surveyed (RGC and displaced amacrines) was multiplied by 4.78 to estimate the number
of inner nuclear layer (INL) amacrines.
RGCs, displaced amacrines, and INL amacrines were counted in 33 fields (200 X, 8 eyes, 6 mice, R26floxGFP reporter). Horizontal cells were counted in 58 sections (8 eyes, 6 mice,
R26floxGFP reporter). All other cell types were counted in 50 to 70 fields (200 X, 16 eyes, 12 mice, Z/AP reporter). Math5+ descendants detected using the Cre lineage system
comprise 2.9% of the adult retina (f ).
TOTAL
Mller glia
bipolar
rod
1,515
cone
1,198
700
RGC
INL
(a)
CELL TYPE
Math5 lineage
cells counted
Table III-1. Cell type distribution of Math5 lineage descendants in wild-type Math5 >Cre transgenic retinas
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Figure III-S2. Birthdating curves for cones (top graphs) and rods (bottom graphs)
in (A) wild-type Math5>Cre; Z/AP and (B) mutant Math5>Cre; R26floxGFP mice
between E14.5 and E17.5 (see Table III-S4). Single BrdU injections were given to
pregnant dams on the indicated days, and photoreceptors were analyzed in the
adult retinas. Curves show the percentage of each photoreceptor type that is
BrdU+ for the hPLAP+ or GFP+ (red), hPLAP or GFP (blue), and combined
(black) progenitor groups. The difference in scale between panels A and B
reflects the relative deficiency of late-born rods (>E17) and relative excess of
early-born cones (<E14) in Math5 -/- mice.
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Figure III-S3. Proneural bHLH factors Neurod1 and Math5 are expressed in
overlapping subsets of progenitor cells during early retinal neurogenesis. E11.5
Math5-lacZ/+ retinal sections were immunostained for gal and Neurod1.
Approximately half of the gal+ cells co-express Neurod1 (arrows, inset). Scale bar,
50 m.
142
cell type
(% of Math5 lineage)
CELL TYPE
(a)
(a/b) x 100
cone
189
21
horizontal
14
amacrine
295
33
INL
187
21
displaced
108
12
358
40
rod
1.6
bipolar
17
1.9
Mller glia
21
2.3
TOTAL
894 (b)
100
All labeled cells were identified and counted in 23 fields (200X, 6 eyes,
Z/AP reporter). All cells in the GCL were assumed to be displaced
amacrine cells, as no cells in Math5 mutant retinas were positive for RGC
markers Brn3a or Brn3b.
143
144
334
257
330
257
1178
rods
cones
INL amacrines
GCL neurons*
TOTAL
1313
287
376
274
376
(a)
total hPLAP
1331
284
368
289
390
(g)
total GFP
80
82
80
84
77
d
a+gd
SEM
concordance (%)#
Standard error of the mean (SEM) calculated from concordance for n = 4 eyes. There was no significant difference among cell
types (one-way ANOVA, P > 0.15).
The incomplete concordance (<100%) in this experiment validates our assumption that Cre levels are not saturated.
There was no significant difference in the number of cells labeled by each reporter in the fields surveyed (paired t test, P > 0.1).
All single and double labeled cells were counted in 18 fields (200X) representing 4 eyes from 3 Math5>Cre; Z/AP; R26floxGFP
mice. hPLAP, alkaline phosphatase; GFP, green fluorescent protein.
(d)
cell type
hPLAP+ GFP+
cells counted
Table III-S2. Dual reporter concordance for Math5>Cre labeled retinal cells
BrdU+
total
rods
70
70
100
cones
53
53
100
INL neurons
62
65
95
GCL neurons
53
53
100
238
241
99
TOTAL
145
% BrdU+
E15.5
E16.5
E17.5
hPLAP+
hPLAP
combined
hPLAP+
hPLAP
BrdU+
136 (12.1)
314 (27.9)
450 (40.0)
64 (0.2)
BrdU
243 (21.6)
431 (38.4)
674 (60.0)
315 (0.8)
38,700* (97.8)
39,100* (98.7)
total
379 (33.7)
684 (66.3)
1,124 (100.0)
379 (1.0)
39,200* (98.9)
39,600* (100.0)
455
(1.1)
combined
519
(1.3)
BrdU+
79 (8.3)
141 (14.5)
220 (22.7)
140 (0.4)
BrdU
214 (22.0)
537 (55.3)
751 (77.3)
317 (0.9)
32,600* (55.3)
33,000* (96.5)
total
293 (30.2)
678 (69.8)
971 (100.0)
457 (1.3)
33,700* (69.8)
34,000* (100.0)
1,073
(3.1)
1,213
(3.5)
BrdU+
28 (1.6)
100 (5.7)
128 (7.3)
383 (0.6)
BrdU
513 (29.1)
1,124 (63.6)
1,637 (92.7)
480 (0.8)
57,400* (92.4)
57,900* (93.2)
total
541 (30.7)
1,224 (69.3)
1,765 (100.0)
863 (1.4)
61,300* (98.6)
62,100* (100.0)
3,881
(6.2)
4,264
(6.8)
BrdU+
2 (0.2)
20 (1.9)
22 (2.1)
122 (0.3)
BrdU
300 (28.5)
732 (69.4)
1,032 (97.9)
264 (0.7)
34,000* (91.6)
34,300* (92.3)
total
302 (28.7)
752 (71.3)
1,054 (100.0)
386 (1.0)
36,700* (99.0)
37,100* (100.0)
2,729
(7.4)
2,851
(7.7)
For the E14.5 pulse, 16 fields (200X) were counted (3 eyes, 3 mice). For the E15.5 pulse, 14 fields (200X) were counted (4 eyes, 2
mice). For the E16.5 pulse, 24 fields (200X) were counted (5 eyes, 4 mice). For the E17.5 pulse, 15 fields (200X) were counted (4 eyes,
3 mice).
* Combined rod totals were estimated by multiplying the combined cone counts by 35.2 (Jeon et al. 1998).
E15.5
E16.5
E17.5
GFP+
GFP
combined
GFP
BrdU+
21 (3)
113 (17)
134 (20)
26 (0.3)
BrdU
87* (13)
455* (67)
542* (80)
83* (1)
7,699* (95)
7,782* (96)
total
108 (16)
568* (84)
676* (100)
109 (1.3)
8,025* (99)
8,134* (100)
BrdU+
8 (1)
97 (16)
105 (17)
32 (0.4)
1,066 (14)
1,098 (15)
BrdU
27* (4)
490* (79)
517* (83)
40* (0.5)
6,337* (85)
6,377* (85)
total
35 (6)
587* (94)
622* (100)
72 (1.0)
7,403* (99)
7,475* (100)
(3)
10 (1)
1,054* (95)
1,068* (96)
58* (0.4)
12,548* (94)
12,606* (94)
total
24 (2)
1,087* (98)
1,111* (100)
123 (0.9)
13,240* (99)
13,363* (100)
1,576 (18)
(4)
65 (0.5)
692 (5)
757
(4)
14* (1)
30
(4)
352
BrdU+
25 (3)
43
(4)
BrdU
(1)
33
326
combined
(6)
BrdU+
115 (1)
1,461 (16)
BrdU
73* (10)
639* (86)
712* (96)
138* (2)
7,212* (81)
7,350* (82)
total
78 (11)
664* (89)
742* (100)
253 (3)
8,673* (97)
8,926* (100)
For the E14.5 pulse, 14 fields (200X) were counted (2 mice). For the E15.5 pulse, 7 fields (200X) were counted (2 mice). For the E16.5
pulse, 9 fields (200X) were counted (2 mice). For the E17.5 pulse, 10 fields (200X) were counted (2 mice).
* Combined rod and cone totals were estimated by multiplying the measured number of inner nuclear layer nuclei by 0.326 for cones or
3.92 for rods. These ratios were determined empirically in Math5 -/- mice (data not shown).
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CHAPTER IV
DYNAMIC EXPRESSION OF GANGLION CELL MARKERS IN RETINAL
PROGENITORS DURING THE TERMINAL CELL CYCLE
Abstract
The vertebrate neural retina contains seven major cell types, which arise
from a common multipotent progenitor pool. During neurogenesis, these cells
stop cycling, commit to a single fate, and differentiate. The mechanism and
order of these steps remain unclear. The first-born type of retinal neurons,
ganglion cells (RGCs), develop through the actions of Math5 (Atoh7), Brn3b
(Pou4f2) and Islet1 (Isl1) factors, whereas inhibitory amacrine and horizontal
precursors require Ptf1a for differentiation. We have examined the link between
these markers, and the timing of their expression during the terminal cell cycle,
by nucleoside pulse-chase analysis in the mouse retina. We show that G2 phase
lasts 1-2 hours at embryonic (E) 13.5 and E15.5 stages. Surprisingly, we found
that cells expressing Brn3b and/or Isl1 were frequently co-labeled with EdU after
a short chase (<1 hr) in early embryos (<E14), indicating that these factors,
which mark committed RGCs, can be expressed during S or G2 phases.
However, during late development (>E15), Brn3b and Isl1 were exclusively
expressed in post-mitotic cells, even as new RGCs are still generated. In
contrast, Ptf1a and amacrine marker AP2 were detected only after terminal
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Introduction
The vertebrate neural retina is populated by seven major cell types, which
are generated in an invariant histotypic order from a common progenitor pool
(Livesey and Cepko, 2001; Wong and Rapaport, 2009). Individual cell fates are
specified by intrinsic transcriptional programs and the retinal microenvironment.
At the onset of retinal neurogenesis, on embryonic day 11 (E11) in the mouse,
the first neurons exit the cell cycle and differentiate as retinal ganglion cells
(RGCs), whose axons form the optic nerves. Mouse RGC birthdates extend from
E11 to P0 with a peak at E14 (Drager, 1985; Young, 1985a). This temporal
profile significantly overlaps the distribution of birthdates for cone photoreceptor,
horizontal and amacrine cells. Rod photoreceptors, bipolar cells and Mller glia
generally have later birthdates.
RGC development is controlled by a complex transcriptional network.
Math5 (Atoh7) is expressed transiently in multipotent precursors (Brzezinski et
al., 2012; Yang et al., 2003) and is necessary for RGC fate specification (Brown
et al., 2001; Wang et al., 2001). The Brn3b (Pou4f2) and Islet1 (Isl1)
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homeodomain genes form two regulatory nodes that are downstream of Math5 in
the RGC differentiation hierarchy (Mu et al., 2008; Pan et al., 2008). Both factors
are thought to be expressed in newly post-mitotic cells, and are abundant in
differentiated ganglion cells (Pan et al., 2008; Pan et al., 2005; Xiang, 1998).
Brn3b appears to mark committed RGC precursors, as it is expressed exclusively
in RGCs and required for terminal differentiation (Erkman et al., 1996; Gan et al.,
1996; Qiu et al., 2008; Xiang, 1998). Isl1 is also required for RGC development
(Mu et al., 2008; Pan et al., 2008), but is expressed in a wider population of cells
(Elshatory et al., 2007a). The Brn3b+ population is essentially contained within
the Isl1 lineage (Pan et al., 2008). Amacrine and horizontal neurons are
specified in part by Ptf1a, a transiently expressed factor that acts downstream of
FoxN4 (Fujitani et al., 2006; Li et al., 2004). Brn3b and Ptf1a are likely to
assemble in opposing transcriptional complexes that regulate RGC or
amacrine/horizontal cell differentiation, respectively (Fujitani et al., 2006; Qiu et
al., 2008).
The sequential birth order of different retinal cell types reflects a shift in
the fate bias of progenitors during development (Livesey and Cepko, 2001). In
addition, the progenitor cell cycle length increases progressively (Alexiades and
Cepko, 1996; Sinitsina, 1971; Young, 1985b). Because neurons do not divide,
and differentiation occurs after the final division, it is generally thought that cell
cycle exit is strictly coupled to fate specification. However, it remains unclear
how cycle length, terminal division, and neurogenic fate are linked, and precisely
when fate commitment occurs (Dyer and Cepko, 2001b; Ohnuma and Harris,
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150
defined by the first chase period in we detected cells positive for the particular
marker and EdU/BrdU.
Histology
Embryonic eyes were fixed for 0.5-1 hour in 2-4% paraformaldehyde
(PFA) at 22C, cryoprotected with 30% sucrose in phosphate-buffered saline
(PBS), and frozen in OCT compound (Tissue-Tek, Torrance, CA). Short fixation
was critical for immunodetection of Brn3b and cyclinD1. Cryosections (10 m)
were blocked with 10% normal donkey serum (NDS) and 1% bovine serum
albumin (BSA) in PBTx (0.1 M NaPO4 pH 7.3, 0.5% Triton X-100) for 4 hours at
22C. Sections were incubated overnight at 4C with primary antisera diluted in
PBTx with 3% NDS, 1% BSA. Sections were then washed in PBS 0.5% BSA,
incubated for 2 hours at 22C with Dylight 488- (1:1000), Dylight 549- (1:1000), or
Dylight 649- (1:500) conjugated secondary antibodies (Jackson
Immunoresearch, West Grove, PA), mounted in Prolong Gold Antifade media
(Invitrogen, Carlsbad, CA), and imaged using the Zeiss LSM510 Meta confocal
microscope at the University of Michigan Microscopy and Image Analysis Core
Facility (MIL).
The primary antibodies were: mouse anti-AP2 (1:1000, 3B5, DSHB,
Iowa City, Iowa); rat anti-BrdU (1:100, BU1/75, Harlan Seralab, Indianapolis, IN);
rabbit anti-gal (1:5000, ICN Cappel, Aurora, OH); rat anti-gal (1:500) (Saul et
al., 2008); mouse anti-Brn3a (1:50, 14A6, Santa Cruz Biotechnology, Santa
Cruz, CA); goat anti-Brn3b (1:200, sc31987, Santa Cruz); mouse anti-cyclin D1
(1:100, A-12, Santa Cruz); chicken anti-GFP (1:2000, Abcam, Cambridge, MA);
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152
153
Results
G2 phase is 1-2 hours long in most retinal cells
The eukaryotic cell cycle is anchored by M phase, when cytokinesis
occurs, and S phase when DNA synthesis occurs. These are separated by G1
and G2 gap phases, respectively (Fig. IV-1A) (Nurse, 2000). As a first step to
evaluate the timing of the transcription factor expression relative to cell cycle exit,
we systematically determined the onset of M phase in cycling progenitors at
different developmental stages using EdU or BrdU pulse-chase experiments. In
this experimental paradigm, cycling progenitors incorporate nucleoside analogs
EdU or BrdU during DNA replication (S phase), and these labeled progenitors
are then followed as they progress through G2 and M phases (Fig. IV-1A). To
assess the onset of M phase, we co-stained embryonic sections for
phosphohistone H3 (PH3) and EdU or BrdU to identify the shortest chase that
yielded double-labeled cells. Histone H3 is transiently phosphorylated for the
entire duration of M phase (Bradbury, 1992) No EdU+ PH3+ cells were detected
after a 1 hr EdU chase, while most PH3+ had punctate EdU labeling after a 2 hr
chase (Fig. IV-1B). These results suggest that M phase initiates at 1-2 hrs after
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the end of S phase, in the majority of progenitors. Similar results were observed
at E15.5 (Fig. IV-1C), and are consistent with measurements of G2 length at
other embryonic times (Sinitsina, 1971; Young, 1985b).
Expression of Brn3b and Isl1, but not Ptf1a or AP2, prior to cell cycle exit
Using the pulse-chase paradigm, it is possible to unambiguously identify
the onset of expression relative to the end of S phase. Previous lineage analysis
and marker co-staining have shown that Brn3b is restricted to RGCs (Badea et
al., 2009; Qiu et al., 2008; Xiang et al., 1995), and that Ptf1a is confined to
inhibitory horizontal and amacrine cells (Fujitani et al., 2006). As a first step in
this analysis, we tested whether Brn3b and Ptf1a truly mark mutually exclusive
populations of committed precursors during early retinal development, by costaining sections from E13.5 retinas for Brn3b, Ptf1a, and the amacrine marker
AP2 (Bassett et al., 2007). We observed no overlap of Ptf1a or AP2 with
Brn3b (Fig 2A) consistent with previous results observed during late gestation
(Bassett et al., 2007; Fujitani et al., 2006; Qiu et al., 2008). As expected, many
AP2-expressing cells were contained within the Ptf1a population, and are
presumed to be precursors of inhibitory amacrine neurons.
We next determined the onset of Brn3b, Ptf1a and AP2 with respect to
the terminal cell cycle as outlined (Fig. IV-1A). At both E13.5 and E15.5, we
observed no overlap between Ptf1a or AP2 and EdU after a 4 hr chase (Fig. IV2B,C), but Ptf1a+ EdU+ cells were apparent after a 12 hr chase (Fig. IV-2D,E).
Although Ptf1a is transiently expressed and vital for development of horizontal
and amacrine cells, its actions are apparently delayed until after cell cycle exit,
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most likely during cell migration. In contrast, at E13.5, we found that Brn3b+
EdU+ cells were present after a 1 hr chase (2-5 per high-power field, Fig. IV-3A).
All Brn3b+ cells were cyclinD1 (cycD1), including the EdU+ cohort, whereas the
vast majority of EdU+ cells (>95%) were cycD1+ (Fig. IV-3A). In most cycling
cells, cycD1 is made during G1 and down-regulated during S phase, and it
controls the G1/S transition. CycD1 is reactivated in G2 (Stacey, 2003), except
in terminally mitotic cells (Yang et al., 2006). In the developing retina, cycD1 can
be detected in all cells, except for post-mitotic neurons (G0) and a small
subpopulation of G2 cells that are likely represent the neurogenic cohort (Barton
and Levine, 2008). Accordingly, we interpret the Brn3b+ EdU+ cycD1precursors at E13.5 as neurogenic cells in late S or G2 phase of the terminal
division. Consistent with interpretation, we observed rare Brn3b+ cells at the
scleral margin (apex), in M phase (PH3+, arrows) or in adjacent pairs
(arrowheads) suggestive of a recent division (Fig. IV-3B). Furthermore, Brn3b+
EdU+ cells can be followed through the cell cycle, with symmetric Brn3b
segregation (Fig. IV-3B), and migration into the GCL with a 12 hr chase at E13.5
(Fig. IV-3C). Many of the resulting Brn3b+ EdU+ cells are in close proximity, as
presumptive daughter pairs. At E15.5, essentially no Brn3b+ EdU+ cells were
observed after 1- or 4-hr chase periods, but were readily detected throughout the
retina after a 12 hr chase (1-4 per high-power field, Fig. IV-3D). Although new
Brn3b+ cells continue to be generated throughout the retina, Brn3b is expressed
exclusively in post-mitotic cells at this stage. Quantitative analysis revealed that
only 4.8 0.9% of Brn3b+ cells at E13.5, and 1.2 0.6% at E15.5, were EdU+
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after a 12 hr chase (Fig. IV-3E). These cells represented the sum of neurogenic
cells initiating Brn3b expression during S/G2 and G0 phase. To estimate the
fraction of Brn3b+ cells at E13.5 that initiate expression during S/G2, we
normalized the Brn3b+EdU+ fractions observed after a 0.5-4 hr chase to the 12
hr chase value (Fig. IV-3E). This analysis revealed that 50-60% of new Brn3b+
cells (accounting for mitotic doubling) initiated expression prior to cell cycle exit
(<4 hr chase).
To confirm these observations regarding the timing of RGC marker
expression relative to terminal M phase, we evaluated a second regulator of
RGC differentiation, Islet-1 (Isl1). We co-stained sections for Brn3b, Isl1 and
EdU following a 30 min chase. We observed numerous Brn3b+ Isl1+ cells that
were EdU+ at E11.5 or E13.5 (Fig. IV-4A,B), whereas no Brn3b+ Isl1+ cells were
EdU+ at E16.0 (Fig. IV-4C). These results indicate that at least two key
regulators of RGC fate are expressed prior to cell cycle exit during early (<E14),
but not during late (>E15), developmental stages.
RGC fate symmetry in marked clones
If progenitors express Brn3b and Isl1 before terminal M phase, they
should give rise to paired RGCs. To test this hypothesis, we performed a clonal
analysis. Retinal explants from E12.5 or E13.5 embryos were infected with
MSCV-IRES-GFP (MIG) retrovirus at low density to generate independent
clones. After culturing 3 days in vitro (DIV), we scored the resulting GFP+ clones
for their size and number of RGCs, using Brn3a or 3b immunoreactivity and
cellular morphology to identify ganglion cells (Fig. IV-5A). The Brn3a paralog is
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functionally interchangeable with Brn3b at the protein level (Pan et al., 2005) and
its spatiotemporal expression pattern is highly overlapping with Brn3b and Brn3c
(Xiang et al., 1995; Xiang et al., 1993).
In 12 explanted retinas, we observed approximately 250 GFP+ clones,
which were 1-16 cells in size. Fourteen (~5%) included at least one Brn3+ RGC.
Among these informative clones, we observed two-cell clones with paired (Fig.
IV-5B,C) or single (Fig. IV-5D) RGCs. Of the 12 neurogenic divisions that could
unambiguously be scored among 14 clones, six were symmetric, resulting in
paired RGCs (Fig. IV-5E). In principle, paired RGCs could arise if both daughter
cells independently adopt the RGC fate, stochastically or in response to the local
retinal environment. Given the very small number of GFP+ ganglion cells
observed in these explants (n = 24), and the high frequency of symmetric fating (6
of 12 divisions), this pattern is unlikely to reflect independent events paired by
chance (2 > 40, df = 2, P < 0.001 for a Poisson distribution). Instead, this
statistical clustering, and the expression of definitive RGC markers in G2 and
through cytokinesis (Fig. IV-3), strongly argue that many RGCs arise in pairs and
descend from progenitors that commit to the ganglion fate during their final cell
cycle, prior to terminal mitosis.
Discussion
The kinetics of the retinal progenitor cell cycle have been extensively
characterized in rodents and other vertebrate species by window and cumulative
nucleoside labeling, cell counting, and percent labeled mitosis (PLM) methods
158
(Alexiades and Cepko, 1996; Fujita, 1962; Li et al., 2000; Sinitsina, 1971; Young,
1985b). In addition, a variety of nuclear factors responsible for generating
histotypic diversity in the retina have been identified through loss- and gain-offunction genetic analysis, and expression studies (Ohsawa and Kageyama,
2008; Swaroop et al., 2010). However, the basic question of when the cell fate
decision is made relative to the terminal cell cycle has remained largely
unanswered. In this paper, we have integrated expression and cell cycle
analysis to determine the precise time that transcription factors controlling RGC,
amacrine and horizontal fates appear during the last (neurogenic) cell cycle.
RGC fate commitment can occur before cell cycle exit
At early developmental times (<E14), we detected Isl1 and Brn3b in many
cells during late S or G2 phases of the terminal cell cycle (Figs. 3,4), similar to
Math5-lacZ (Brzezinski et al., 2012), and in contrast to previous reports (Pan et
al., 2008; Xiang et al., 1993). This interpretation is based on three convergent
results: the precise timing of PH3 (Fig. IV-1), Brn3b and Isl1 (Fig. IV-4) onset
relative to the end of S phase, the mutually exclusive pattern of cycD1 and Brn3b
expression (Fig. IV-3A), and the presence of Brn3b+ M phase cells (Fig. IV-3B).
There are three possible explanations for the observation of RGC markers
in dividing cells. First, the immunopositive cells may reflect low-level leaky
transcription of Brn3b or Isl1 that is not biologically meaningful. This seems
unlikely given that the expression in S/G2 phase progenitors is often as strong as
that observed in post-mitotic cells (e.g. arrows in Fig 4A,B). Second, Isl1 and
Brn3b may not accurately mark committed RGC precursors. Indeed, Isl1 is also
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Mason, 2010). Alternatively, the relatively early onset of Brn3b expression may
reflect the position of RGCs as the first sampled fate in the histogenetic
sequence (Wong and Rapaport, 2009).
Our clonal data are generally consistent with previous observations of
terminal divisions in the early retina. In rodent and frog lineage analysis, many
two-cell clones have been observed during early development, but typically these
cells have discordant fates (Holt et al., 1988; Turner et al., 1990; Wong and
Rapaport, 2009). Notably, among 114 two-cell clones analyzed in these reports,
only one contained paired RGCs (Holt et al., 1988). During early zebrafish
development, most ath5-expressing progenitors, monitored by time-lapse
imaging of mosaic ath5-GFP embryos, undergo a terminal division and give rise
to at least one ganglion cell (Poggi et al., 2005). The daughters typically have
discordant fates, but paired RGCs may arise from wild-type cells in an ath5
mutant environment.
Similar to our finding of mitotic Brn3b+ cells (Fig. IV-3B), RA4+ cells were
observed in terminal mitotic anaphase in the chick retina (Waid and McLoon,
1995), and migrating in pairs shortly after mitosis (McLoon and Barnes, 1989).
Although originally interpreted as RGC precursors, it is no longer clear that the
RA4 neurofilament antigen marks ganglion cells exclusively (Gutierrez et al.,
2011). Beyond these observations, there is precedent for G2 commitment during
neurogenesis of other cell types. In the chick retina, committed horizontal cell
precursors typically arrest in G2 and undergo a final non-apical mitosis (Boije et
al., 2009), which may explain the presence of paired horizontal cell clones of a
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single subtype (Rompani and Cepko, 2008). In the ferret cerebral cortex,
progenitors lose their competence to respond to environmental signals following
terminal G2 phase (McConnell and Kaznowski, 1991). Similarly, heterochronic
analysis of dissociated rat retinal cells suggests that progenitors commit to the
amacrine fate (VC1.1+) during G2/M of the last cell cycle (Belliveau and Cepko,
1999).
Post-mitotic fate plasticity in the retina
In this study, the amacrine marker Ptf1a was only detected in post-mitotic
cells, at both E13.5 and E15.5 (Fig. IV-2B-E). In Ptf1a mutant mice carrying Cre
knock-in alleles, many lineage-marked cells expressed Brn3b and adopted RGC
fates (Fujitani et al., 2006). Taken together, these results suggest a degree of
post-mitotic plasticity, such that some amacrine cells solidify their identity after
the terminal division. Likewise, photoreceptor and bipolar precursors retain the
ability to switch fates after cell cycle exit (Adler and Hatlee, 1989; Brzezinski et
al., 2010; Ng et al., 2011; Oh et al., 2007). Indeed, the bZIP factor NRL, which
instructs rod photoreceptor fate, initiates expression soon after terminal M phase,
throughout development (Akimoto et al., 2006); data not shown).
We observed a gradual restriction in the expression of Brn3b and Isl1, as
well as Math5-lacZ (Brzezinski et al., 2012), to post-mitotic cells during late
gestation (Fig. IV-3D). At these ages (>E15), many RGCs are still generated,
including migrating EdU+ Brn3b+ cells (Drager, 1985; Young, 1985a). In the
early (<E14) retina, we observed both symmetric and asymmetric patterns of
RGC marker expression among daughter cells in clones, suggesting that some
162
cells commit to the RGC fate after terminal mitosis in the early retina (Fig. IV-5E)
(Brzezinski et al., 2012). The difference in the timing of RGC marker onset
between the early and late retina cannot be explained by a change in the length
of G2, which is relatively static (Fig. IV-1), or by the progressive lengthening of
the cell cycle during development (Alexiades and Cepko, 1996; Sinitsina, 1971;
Young, 1985b). Indeed, if transcription initiates after G1 or S phase with a fixed
time delay, then the onset of expression following terminal S phase should be
advanced (closer to S), rather than delayed as we observed during the course of
development. It is more likely that progenitor fate decisions occur before or after
cell cycle exit, depending on the lineage or developmental stage.
What could explain these shifts in expression kinetics? Environmental
signals elaborated by differentiating neurons, such as Delta ligand or Shh,
increase as RGCs accumulate (Ahmad et al., 1997; Wang et al., 2005; Yang,
2004; Yu et al., 2006) and could delay the onset of RGC transcription factor
expression. Alternatively, the onset may be intrinsically programmed to shift over
time, and is potentially regulated by the same upstream cascades that control
cell cycle exit. Further studies are needed to distinguish these mechanisms.
Nonetheless, it is clear that cell fate determination and cell cycle exit, though
correlated, are not strictly causally related events.
Acknowledgements
The authors are grateful to Dellaney Rudolph and Melinda Nagy for technical
support; to Helena Edlund for Ptf1a antisera; to Chris Edwards, and the UM
microscopy and image analysis laboratory staff, for technical advice; to Sean
163
Morrison for the MIG retroviral construct; to Joe Brzezinski, Nadean Brown, Chris
Chou, Sally Camper and David Turner for valuable discussions and critical
reading of the manuscript. This research was funded by National Institutes of
Health (NIH) R01 grant EY14259 (TG). LP was supported by NIH T32 grants
EY13934 and GM07863.
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Figure IV-1. Timing of cell cycle progression in the mouse retinal neuroepithelium
at E13.5 and E15.5. (A) EdU pulse-chase experiments. Phosphohistone H3 (PH3)
is expressed during M phase, while cyclin D1 is primarily expressed during G1 and
early S phases. After an EdU pulse, labeled S phase cells progress into G2, M
and G1/G0 phases. (B) Diagram of interkinetic nuclear migration, showing the
positions of progenitor nuclei at each stage of the cell cycle. Following terminal M
phase, G0 daughters segregate vertically according to their cell fate. Ganglion
cells (rgc) migrate toward the base (vitread), while photoreceptors (pr) migrate
toward the apex (sclerad). (C-D) E13.5 and E15.5 embryos stained for PH3 and
EdU or BrdU following a 1-2 hour chase. No PH3+ cells are EdU+ after a 1 hour
chase, but most are EdU+ or BrdU+ after a 2 hour chase. Therefore, M phase
initiates at 1-2 hours after S phase in most cells, at both of developmental stages.
nbl, neuroblastic layer; gcl, ganglion cell layer. Scale bar, 50 m.
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166
167
Figure IV-3. The onset of Brn3b and Isl1 expression within individual cells is
progressively delayed during retinal development. (A) Sections from E13.5
embryos co-stained for Brn3b, cyclinD1 (cycD1) and EdU following a 1 hr chase.
In some cells, Brn3b is co-localized with EdU (arrows), but not with the G1/earlyS phase marker cycD1, indicating that Brn3b expression initiates in late S or G2
phase. Insets show two cells that are Brn3b+ EdU+ cyclinD1. One has a
punctate EdU staining pattern, indicating incorporation in late-replicating DNA
regions at the end of S phase. (B) Sections from E13.5 embryos stained for
Brn3b and the mitotic marker PH3 or the nuclear counterstain DAPI. Brn3b+
cells in M phase (arrows) and presumptive daughter pairs (arrowheads) are
indicated. (C-D) Sections from E13.5 or E15.5 embryos co-stained for Brn3b and
EdU following the indicated chase period. At E13.5 (C), many newly generated
Brn3b+ cells are co-labeled with EdU after a 2, 4 or 12 hr chase. By 12 hrs,
some EdU-labeled cells migrate to the nascent GCL, and many Brn3b+ EdU+
cells are in close proximity as presumptive daughter pairs. At E15.5 (D), no
Brn3b+ EdU+ cells are observed after 1 hr chase, and very few are detected
after a 4 hr chase, but Brn3b+ EdU+ are readily detected after a 12 hr chase
(arrows). (E) Quantitative analysis of Brn3b and EdU co-labeling. The
abundance of co-labeled cells is plotted as a percentage of all Brn3b+ cells (left
scale) or neurogenic Brn3b+ cells (right scale, normalized to the 12 hr chase
value at E13.5). Approximately 30% of newly Brn3b+ cells initiated expression
prior to cell cycle exit at E13.5 (gray line). In contrast, few Brn3b+ cells are
co-labeled with EdU prior to 12 hrs at E15.5, indicating that RGCs are specified
later at this developmental stage. Scale bar, 50 m.
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Figure IV-4. Co-expression of Brn3b and Isl1 during or shortly after the
terminal cell cycle. (A-C) Sections from E11.5, E13.5, and E16.0
embryos co-stained for Brn3b, Isl1 and EdU following a 30 min chase.
At E11.5 (A) and E13.5 (B), multiple Brn3b+ Isl1+ EdU+ cells are
observed (arrows), indicating that RGC markers can initiate expression
prior to cell cycle exit, in late S or G2 phase. In contrast, at E16.0 (C), all
Brn3b+ Isl1+ cells are EdU (examples marked by arrowheads in D-F). The
insets show orthogonal Z-stack views of magnified Brn3b+ Isl1+ EdU+ cells.
Scale bar, 50 m.
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Figure IV-5. Paired ganglion cells can be generated from retinal progenitors by
symmetric terminal division. (A) Retinas were explanted from E12.5 or E13.5
embryos, infected at clonal density with MSCV-IRES-GFP (MIG) retrovirus
(green), and cultured for 3 days in vitro (DIV). The micrograph shows a crosssection from a representative explant (bracket) coimmunostained for Brn3a
(magenta) and GFP (green). The schema shows informative two-cell GFP+
clones: a symmetric [S] clone containing two Brn3+ RGCs, and an asymmetric
[A] clone with one Brn3+ RGC. (B-D) Confocal Z-stack projections and drawings
from representative symmetric (B, C) or asymmetric (D) clones containing RGCs.
Most Brn3+ cells have long processes (arrows), confirming that they are
differentiated RGCs. (E) Summary of observed clones containing at least one
Brn3+ cell. In these experiments, RGCs were identified using Brn3a (12 clones)
or Brn3b (2 clones) antisera interchangeably. Among 12 informative divisions,
an equal number with symmetric and asymmetric fates was observed. Scale
bars, 10 m in A; 5 m in B-D.
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CHAPTER V
Abstract
The basic-helix-loop helix factor Math5 (Atoh7) is required for retinal
ganglion cell (RGC) development. However, only 10% of Math5-expressing cells
adopt the RGC fate, and most become photoreceptors. In principle, Math5 may
actively bias progenitors towards RGC fate or passively confer competence to
respond to instructive factors. To distinguish these mechanisms, we
misexpressed Math5 in a wide population of precursors using a Crx BAC or 2.4
kb promoter, and followed cell fates with Cre recombinase. In mice, the Crx
cone-rod homeobox gene and Math5 are expressed shortly after cell cycle exit,
in temporally distinct, but overlapping populations of neurogenic cells that give
rise to 85% and 3% of the adult retina, respectively. The Crx > Math5 transgenes
did not stimulate RGC fate or alter the timing of RGC births. Likewise, retroviral
Math5 overexpression in retinal explants did not bias progenitors towards the
RGC fate or induce cell cycle exit. The Crx>Math5 transgene did reduce the
abundance of early-born (E15.5) photoreceptors two-fold, suggesting a limited
cell fate shift. Nonetheless, retinal histology was grossly normal, despite
widespread persistent Math5 expression. In an RGC-deficient (Math5 knockout)
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period, from E12.5 to P10 (Carter-Dawson and LaVail, 1979; Swaroop et al.,
2010), because rods compose ~80% of the mature retina (Jeon et al., 1998).
Heterochronic co-culture and transplantation experiments, in which early
embryonic and late RPCs were cultured in unequal ratios, have suggested that
fate determination is largely a cell intrinsic process (Belliveau and Cepko, 1999;
Rapaport et al., 2001; Reh, 1992; Watanabe and Raff, 1990). Indeed,
progenitors cultured at low density can develop into each of the major cell
classes, with similar diversity and proportions as the intact retina, in the absence
of environmental feedback signals (Adler and Hatlee, 1989; Cayouette et al.,
2003; Reh and Kljavin, 1989). However, extrinsic signals can influence
progenitor cell cycle dynamics and override fate decisions in vivo (Cepko, 1999;
Ezzeddine et al., 1997; Kim et al., 2005; Yang, 2004). Collectively, these
observations are consistent with a temporal, or serial, competence model for
retinal development (Cepko et al., 1996; Livesey and Cepko, 2001; Reh and
Cagan, 1994; Wong and Rapaport, 2009). According to this model, RPCs pass
through discrete competence states over time, in which they can adopt a limited
number of cell fates. Within each state, the decision to exit the cell cycle and the
final histotypic choice are influenced by extrinsic signals.
Two prototypical intrinsic factors important for development of mouse
RPCs into specific types of neurons are the cone-rod homeodomain (HD) factor
Crx and the basic-helix-loop-helix (bHLH) factor Math5 (atonal homolog Atoh7).
Crx, and closely related factor Otx2, are expressed in during or shortly after the
terminal cell cycle in tripotential precursors that give rise to photoreceptors and
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bipolar cells (Furukawa et al., 1997; Muranishi et al., 2011). The precise role of
Crx remains unclear. In mice, Crx expression initiates at E12.5 and is necessary
for proper development of photoreceptors, and may be partially redundant with
Otx2 in conferring competence for photoreceptor specification (Chen et al., 1997;
Furukawa et al., 1999; Nishida et al., 2003; Sato et al., 2007). Crx works in
concert with other transcription factor to regulate photoreceptor gene expression
(reviewed in (Hennig et al., 2008; Swaroop et al., 2010)), and Crx is abundant in
adult rods, cones and bipolar cells. The 5 regulatory DNA for Crx has been
extensively characterized, and a critical 2.4 kb promoter region is thought to
faithfully recapitulate the endogenous Crx pattern. This segment has been used
to drive Cre, lacZ, and regulators of rod photoreceptor specification in transgenic
mice (Cheng et al., 2006; Furukawa et al., 2002; Koike et al., 2005; Nishida et al.,
2003; Oh et al., 2007).
Like Crx, the role of Math5 in fate specification has not been fully
elucidated. Math5 (Atoh7) is a single-exon gene that is transcribed by retinal
progenitors in a spatiotemporal pattern that mirrors RGC births (Brown et al.,
1998; Brzezinski et al., 2012; Prasov et al., 2010). This factor is transiently
expressed by RPCs during or after their terminal cell cycle (Brzezinski et al.,
2012; Feng et al., 2010; Kiyama et al., 2011) and is required for RGC
development. Math5 mutant mice have very few RGCs (<5%) and lack optic
nerves (Brown et al., 2001; Wang et al., 2001). Apart from the deficiency of
RGCs, all other retinal cell classes are preserved (Brown et al., 2001; Brzezinski
et al., 2005). The mRNA profiles of Math5 mutant retinas are altered, with
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polyA signal. The mouse Crx promoter and proximal regulatory region were
amplified by PCR and inserted upstream as a 2.4 kb XhoI-SalI fragment
(Furukawa et al., 2002; Oh et al., 2007). A matched Crx>Cre transgene was
then generated from the Crx>Math5-IRES-Cre plasmid by precise deletion of
Math5 and IRES sequences using the single-strand oligonucleotide (ss oligo)
recombineering method (Thomason et al., 2007), with a 70 nt antisense oligo
(Suppl. Table 1) and AscI selection.
To faithfully express Math5 in the endogenous Crx pattern, we generated
BAC transgenes by RED recombineering (Lee et al., 2001). The targeting
construct was assembled, with short (400 bp) 5 and 3 homology arms (H)
flanking a Math5-IRES-Cre-FRT-amp-FRT cassette. This was equivalent to the
conventional transgene, but included an FRT-amp-FRT selection cassette (Gene
Bridges, Heidelberg) downstream of the SV40 polyA signal. The 5 homology
arm extends from Crx intron 1 to the exon 2 initiation (ATG) codon, while the 3
homology arm contains sequence from Crx intron 2. A matched control (CreFRT-amp-FRT) was then generated by ss oligo recombineering, with the 70 nt
antisense oligo (Suppl Table 1) and AscI selection.
Linearized targeting plasmids were used in parallel to target mouse BAC
clone RP23-81H17 by RED-mediated homologous recombination in strain
SW105 (Warming et al., 2005) after heat induction. This 219 kb BAC contains
134 kb 5 and 69 kb 3 DNA flanking the Crx gene. Targeted BAC clones were
selected on ampicillin and chloramphenicol plates at 30C, and verified by
junctional PCR and DNA sequencing. The amp seletion cassette was then
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1, and two reverse primers (Suppl. Table 1). Dual products were closely
matched for size and G+C content, and analyzed using a 3730XL capillary
electrophoresis unit (Applied Biosystems, Carlsbad, CA) and Gene Marker
software (SoftGenetics, State College, PA).
Quantitative RT-PCRs were performed using custom Taqman probes and
Universal Taqman Mastermix (Applied Biosystems), and were analyzed on the
ABI 7600 Real Time PCR System. Critical cycle threshold levels were
normalized to Gapdh internal controls, using two-fluorophore (VIC and FAM)
detection. Fold activity was calculated using the ddCt method (Livak and
Schmittgen, 2001) and reported relative to the Crx>Math5 BAC expression level.
Expression copy-number levels were calculated for conventional and BAC
transgenes by normalizing to endogenous Crx values, using the mean ratio
determined in triplex competitive RT-PCRs (k). Measurements were obtained
using independent RNA pools from 2-5 mice of each genotype.
Histology
For section immunostaining, eyes or embryonic heads were fixed in 2-4%
paraformaldehyde (PFA) 0.1 M NaPO4 pH 7.3 for 30-60 min at 22C, processed
through a 10-30% graded sucrose series, embedded in OCT (Tissue-Tek,
Torrence, CA) and cryosectioned at 10 m. For flatmount preparations, eyes of
P1 or adult mice were removed and fixed in 4% PFA for 5 min. The optic nerves
were then transected, and the retinas were teased apart from other ocular
tissues, fixed in 4% PFA for 25 min. After immunostaining, retinas were incised
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with 6-8 radial cuts and flattened with the ganglion cell layer (GCL) facing
upward.
For immunodetection, slides or whole retinas were blocked in a solution of
10% normal donkey serum (NDS), 1% bovine serum albumin (BSA) in PBTx (0.1
M NaPO4 pH 7.3 0.5% Triton X-100) for 1-4 hrs. To reduce mouse-on-mouse
background associated with mouse monoclonal primary antibodies, donkey antimouse IgG Fab fragments were added at 0.8 mg/mL to some blocking reactions.
Primary antibodies were applied overnight at 4C and diluted in 3% NDS 1% BSA
in PBTx. Sections or retinas were then washed in PBS, incubated for 2 hrs at
22C with Dylight-conjugated secondary antibodies and 4',6-diamidino-2phenylindole (DAPI), and mounted in Prolong Gold Antifade (Invitrogen, Grand
Island, NY). Slides were imaged using the Zeiss LSM510 Meta confocal system
or an Olympus BX-51 epifluorescence microscope.
The primary antibodies were mouse anti-AP2 (1:1000, DSHB, Iowa City,
IA); rabbit anti-gal (1:5000, ICN Cappel, Aurora, OH); rat anti--galactosidase
(1:500, (Saul et al., 2008)); rat anti-BrdU (BU1/75, 1:100, Harlan Seralab,
Indianapolis, IN); mouse anti-calbindin (CB-955, 1:500, Sigma, St. Louis, MO);
rabbit anti-cleaved-caspase 3 (1:100, Cell Signaling, Beverly, MA); sheep antiChx10 (1:250, Exalpha, Shirley, MA); mouse anti-Cre (clone 7.23, 1:300,
Covance, Princeton, NJ); rabbit anti-Crx (1:1000, (Zhu and Craft, 2000));
chicken anti-GFP (1:2000, Abcam, Cambridge, MA); mouse anti-hPLAP
(monoclonal 8B6, 1:250, Sigma); mouse anti-PKC (MC5, 1:100, Sigma); rabbit
anti-mCar (1:500, Millipore, Billerica, MA); mouse anti-syntaxin (HPC-1, 1:1000,
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the level of Crx transgene (Cre) expression among progenitors giving rise to
different cell types, we conducted dual reporter concordance experiments, as
described (Brzezinski et al., 2012). Coexpression of GFP and hPLAP was
scored in the outer nuclear (ONL), inner nuclear (INL) and ganglion cell (GCL)
layers of adult triple transgenic mice (Crx>Math5 BAC or Crx>Cre BAC; Z/AP;
R26floxGFP).
Quantitative assessment of RGCs in transgenic mice
Retinal ganglion cells were counted in Crx>Math5 Tg and non-transgenic
littermates at P0 and P22, using Brn3a or retrograde axon labeling to mark
RGCs. The fractional contribution of RGCs to the GCL (DAPI nuclei) was
determined from 20 sections (400X) representing n = 2 eyes of each P22
genotype, and 12-22 sections (200X) representing n = 4-6 eyes for each P0
genotype.
To evaluate transgenic rescue of RGC development in mutants,
Crx>Math5 Tg or Crx>Math5 BAC mice were crossed to Math5 knockout (KO)
mice (Atoh7tm1Gla, (Brown et al., 2001)) for two or more generations. Eyes from
informative embryonic, neonatal and adult littermates were immunostained as
sections or flatmounts. Retinal cell death was assessed at E16.5 using activated
Caspase-3 staining (Gown and Willingham, 2002). RGCs and apoptotic cells
were counted in 18 sections (200X) representing n = 6 eyes of each genotype.
EdU pulse-chase and birthdating analysis
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To evaluate the overlap between Crx+ and Math5+ cell populations, and
compare the timing of Crx and Math5 expression, pregnant dams carrying E13.5
or E15.5 Math5-lacZ/+ embryos were given to a single intraperitoneal injection of
EdU (6.7 g/g body mass). Embryos were harvested after a 4-hr chase and their
retinas were stained for Crx, gal and EdU. For other short-term labeling
experiments, a single pulse of EdU or BrdU was given to pregnant dams 1 hr
prior to harvest.
To assess alterations in the fate distribution of neurogenic cells exiting
mitosis on different days, pregnant dams carrying Crx>Math5 Tg (line 251) and
non-transgenic control embryos were given a single injection of BrdU (100 g/g
body mass) on E12.5, E13.5 or E15.5. Retinal sections from the resulting mice
were stained for BrdU at P21, and the distribution of strongly BrdU+ cells among
GCL, INL and ONL layers was determined. For the E15.5 pulse, we counted 24
sections (200X) from n = 6 eyes of each genotype, representing a total of 816
birthdated cells in Crx>Math5 Tg mice and 1005 birthdated cells in control mice.
To evaluate late-stage RGC births, dams carrying Crx>Math5 BAC (line 60) and
control embryos were pulsed with EdU at E17.5 and harvested at P22. Likewise,
Crx>Math5 Tg (line 251) pups with Math5 KO and heterozygous (het) genotypes,
and non-transgenic littermates, were pulsed with BrdU at P1 and harvested at
P22.
RGC birthdating curves were generated for rescued and control
littermates by giving single EdU pulses at E11 and E12, E13.5, E15.5, or E17.5.
The resulting pups (four genotypes) were harvested at P1 and their retinas were
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stained for Brn3a and EdU as flatmounts. Two 0.05 mm2 areas in the central
retina were imaged as confocal Z-stacks through the GCL for each flatmount
preparation. The density of RGCs (Brn3a+ cells per mm2) and birthdated RGCs
(Brn3a+ EdU+ cells per mm2) was determined by direct counting. The
normalized RGC birth fraction was calculated by dividing the number of RGCs
born at each time point by the sum of RGCs born in all four time points (E11-E12,
E13.5, E15.5, E17.5).
Statistics
Comparisons were made using a two-tailed Students t-test in Microsoft
Excel in cases where equal variance was observed. The Welch t-test was used
for comparisons among groups of unequal variance. Errors are reported for
biological replicates as SDM (standard deviation of the mean) unless otherwise
noted. Jitter plots were generated with Prism software (Graphpad, La Jolla, CA).
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CNTF (10 ng/mL, Peprotech), penicillin (50 U/mL), streptomycin (50 g/mL), and
gentamicin (0.5 g/mL).
The Math5-IRES-GFP retroviral plasmid was constructed by inserting a
Math5 cassette in the bicistronic MSCV-IRES-GFP (MIG) retroviral vector (Van
Parijs et al., 1999). MSCV-IRES-dnMAMLGFP was generated by replacing the
GFP cassette in MIG with dnMAMLGFP. This encodes a fusion protein with
residues 12-72 of mouse MAML1 (mastermind-like) at the N-terminus and GFP
at the C-terminus (Maillard et al., 2004). Retroviral stocks were prepared in
parallel by calcium phosphate transfection of the Phoenix ecotropic packaging
cell line (Pear, 2001; Swift et al., 2001) with plasmid vectors. Polybrene
(hexadimethrine bromide, 0.8 g/mL, Sigma Aldrich, St. Louis, MO) was added to
filtered media containing infectious particles. These viral stocks were titered on
NIH3T3 cells and diluted to ~8 105 CFU (colony forming units) per mL.
Transductions were performed by pipetting one drop (~25 L) on top of each
fresh explant, to sparsely mark dividing cells (Roe et al., 1993) and their
descendants.
Infected explants were cultured for 7 days at the gas-media interface at
37C under 5% CO2. Half of the media was replaced with fresh media on days 2,
4 and 6. After one week in culture, explants were fixed in 4% PFA for 30 min and
processed for cryosectioning. Serial thick (30 m) cryosections were
immunostained for GFP and Brn3a, and imaged as 3-dimensional confocal Zstacks. Clones were scored for size (number of GFP+ cells) and composition
(number of Brn3a+ RGCs). A clone was defined as an isolated group of directy
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apposed GFP+ cells, separated by at least 4 cell bodies from other GFP+ cells.
We scored 3-4 explants per virus, giving a total number of 70 (IRES-GFP), 60
(Math5-IRES-GFP) and 52 (IRES-dnMAML) clones, respectively.
Results
Crx and Math5 are expressed in comparable neurogenic cell populations
Crx and Math5 progenitor cell populations give rise to 85% (rods, cones
and bipolar cells) and 3% of the mouse adult retina (Brzezinski et al., 2012;
Furukawa et al., 1997; Jeon et al., 1998), respectively (Fig. V-1A). To compare
these factors directly, we examined their overlap and onset of expression relative
to the terminal S phase, using a lacZ allele (Atoh7tm1Gla, (Brown et al., 2001)) as a
proxy for Math5. We found that Crx and gal are expressed in distinct, but
overlapping, cohorts of cells at both E13.5 and E15.5 (Fig. V-1B,C), consistent
with the lineage profile of Math5 descendants, over half of which are
photoreceptors (Brzezinski et al., 2012). Using EdU or BrdU pulse-chase
analysis at these time points, we determined that a small number of Crx and gal
double-positive cells at E13.5 and E15.5 were also labeled with EdU after a 4-hr
chase (Fig. V-1). At these developmental stages, four hours is sufficient time for
some cells progress through S, G2, and M phases and enter G0 (Prasov and
Glaser, 2012; Sinitsina, 1971; Young, 1985b). Given that both of these factors
are expressed during or after the terminal division (Brzezinski et al., 2012;
Muranishi et al., 2011); and data not shown), these results suggest that Crx and
Math5 can be made in the same cells, simultaneously or sequentially, at the time
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when cell fate is determined. While Math5 is short-lived, Crx expression persists
in a broad population of differentiated photoreceptor and bipolar cells (Brzezinski
et al., 2012; Furukawa et al., 2002; Furukawa et al., 1997).
Crx>Math5 conventional and BAC overexpression systems
To critically assess the role of Math5 in biasing progenitor cell fate, we
generated transgenic mice with ectopic Math5 expression. Given the overlap of
endogenous Crx and Math5 expression, and the similar nature of these
neurogenic cells, we chose the Crx promoter to broadly express Math5 in a large
population of early post-mitotic precursors. The 2.4 kb Crx promoter has been
extensively characterized and is thought to drive specific expression in
photoreceptor precursors, and mature rods, cones and bipolar cells (Furukawa et
al., 2002). However, to limit position effects and ensure faithful Crx expression,
we also built a BAC transgene containing 134 kb regulatory DNA upstream of the
Crx start site (exon 1), and 69 kb downstream of the polyadenylation signal (exon
3). We then generated conventional and BAC transgenic mice, termed
Crx>Math5 Tg and Crx>Math5 BAC, respectively (Fig. V-2A). The transgenes
express Math5 and Cre from bicistronic transcripts. The Cre recombinase
allowed us to trace the fate of cells expressing transgene-derived Math5 using
the R26floxGFP reporter (Mao et al., 2001), which makes GFP after excision of a
loxP-flanked stop signal. In parallel, we generated matched Crx>Cre and
Crx>Cre BAC control transgenic mice to confirm the Crx lineage, and isolate
Math5 effects.
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related homeodomain factor Otx2 (Bovolenta et al., 1997; Simeone et al., 1993).
In Crx>Cre BAC mice (line 60) and Crx>Math5 BAC mice (line 764), cumulative
transgene expression (GFP) was confined to the retina (Fig. V-2B), and was not
observed in the RPE or ciliary body as noted with the conventional transgenes
(Fig. V-S2). Again, Cre+ cells were co-extensive with the Crx population of cells
at both adult and embryonic time points (Fig. V-2B). Within the adult retina, only
a few scattered cells were labeled with GFP in the inner INL and GCL by the
Crx>Cre BAC and Crx>Math5 BAC transgenes, as expected. Overall, the
patterns of BAC and conventional transgene expression were very different,
suggesting that the Crx 2.4 kb promoter is active beyond the endogenous Crx
domain.
To further characterize the BAC and conventional transgenes, we
assessed the distribution and level of Math5 mRNA expression by RT-PCR. In
the Crx>Math5 Tg, transcripts were evaluated in various tissues using primers
specific to transgenic (Tg) or endogenous Math5 (Fig. V-2B). Both species were
confined largely to the eye, with a low level of endogenous Math5 mRNA in the
brain (Saul et al., 2008). Only transgenic Math5 was detected in the adult retina,
consistent with the known patterns of Math5 and Crx expression (Brown et al.,
1998; Brzezinski et al., 2012; Chen et al., 1997; Furukawa et al., 1997). To
quantitatively compare the levels of BAC and conventional Crx>Math5 transgene
expression, we first determined the ratio of Crx>Math5 BAC and endogenous Crx
transcripts using a triplex competitive RT-PCR assay (Prasov et al., 2010) with a
common end-labeled forward primer located in Crx exon 1 (Fig. V-S3). We found
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R26floxGFP. After a 1 hr EdU pulse, no Cre+ EdU+ cells were detected (Fig. VS7A). However, many EdU+ cells were observed in GFP+ vertical stripes (Fig.
V-S7B), where most labeled Brn3b+ RGCs were localized (Fig. V-S7C). These
findings are consistent with the general clustering of GFP+ cells in the adult
retinas, and suggest a clonal origin (Reese et al., 1999; Turner and Cepko,
1987). Similar patterns were observed for the Crx>Math5 Tg embryos, but the
relative abundance of GFP+ progenitors was a much greater (data not shown).
Crx>Math5 expression does not stimulate RGC genesis
Given the limitations of using the Cre lineage reporter to assess cell fate,
we employed other metrics to assess the effects of broad Math5 overexpression
on the overall fate distribution. We focused primarily on ganglion cells for these
experiments, because Math5 is necessary for RGC development, and we used
Crx>Math5 Tg, because this transgene is expressed at much higher levels than
the BAC counterpart (Fig. V-2D). We observed a similar abundance of Brn3a+ or
rhodamine dextran-labeled RGCs in Crx>Math5 Tg and control mice throughout
development (Fig. V-4A-C). At P0, prior to the neonatal culling of RGCs (Erkman
et al., 2000; Farah and Easter, 2005; Galli-Resta and Ensini, 1996; Young,
1984), 56 5% SD of GCL neurons in Crx>Math5 Tg retinas were RGCs
(Brn3a+), similar to controls (59 5% SD, P = 0.4, Fig. V-4D). Likewise, at P22,
no significant difference was observed in the fraction of RGCs (rhodamine
dextran-labeled) in the GCL between the two genotypes (41.1 0.1% SD for wildtype, 43 8% SD for Crx>Math5 Tg, P = 0.8 Fig. V-4D) or previous reports
(41 4% SD (Jeon et al., 1998)). We conclude that broad ectopic Math5
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expression does not promote RGC fate, alter the survival of RGCs, or drive Brn3
expression in a Math5 wild-type retina.
Crx>Math5 expression alters the distribution of early born cell types
Given the persistent high level of expression of the Crx>Math5 Tg in rods,
cones and bipolar cells, we assessed overall retinal histology in plastic sections
(Fig. V-4E). The morphological features of photoreceptor nuclei, inner and outer
segments, and other layers were not affected at this level. However, subtle fate
shifts might occur during development, which are counterbalanced by
homeostatic feedback mechanisms. To assess these effects, we used a
birthdating approach. Embryos were exposed to single BrdU pulses at E12.5,
E13.5, or E15.5 and their retinas were analyzed at P22. At each time point,
fewer birthdated nuclei were observed in the ONL of Crx>Math5 Tg mice than
controls (Fig. V-5A, Suppl. Fig 8). For quantitative analysis, we focused on the
E15.5 time point, as these litters contained a sufficient number of animals of each
genotype for statistical comparisons. We counted the number of strongly BrdU+
nuclei in ONL, INL and GCL layers. We observed a 2-fold decrease in the
fraction of birthdated photoreceptors (ONL cells) in Crx>Math5 Tg retinas
(21 3% SD) compared to controls (43 2% SD, t-test P < 10-3), with a
corresponding increase in the INL and to lesser extent GCL (Fig. V-5A).
In principle, the loss of early-born photoreceptors could be due to cell
death from persistent Crx>Math5 Tg expression or a small, bona fide fate shift.
To distinguish these mechanisms, we evaluated Crx>Math5 Tg and control
retinas for apoptosis by activated Caspase-3 immunostaining. At multiple time
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points between E13.5 and P0, we observed 1-2 apoptotic cells per field (200X) in
both Crx>Math5 Tg and control retinas (Fig. V-S9), consistent with previous
studies of cell death in the embryonic retina (Vecino et al., 2004). Crx>Math5 Tg
is thus unlikely to induce cell death in photoreceptor precursors. Instead, ectopic
Math5 appears to shift the fates of some early rod and cone photoreceptors.
Crx>Math5 expression does not extend the temporal profile of RGC births
In Crx>Math5 Tg mice, the expression of Math5 is extended through
postnatal development, whereas endogenous Math5 mRNA is downregulated by
P0 (Brown et al., 2001; Brzezinski et al., 2012). To test whether prolonged
expression in neurogenic cells extends the profile of RGC births, we pulsed
Crx>Math5 Tg mice with BrdU at P1 and harvested eyes at P22. In the central
two-thirds of the retina, no BrdU+ cells were detected in the GCL of either
genotype (Fig. V-5B), consistent with the completion of displaced amacrine and
RGC genesis in these areas by P1 (Farah and Easter, 2005; LaVail et al., 1991;
Reese and Colello, 1992; Voinescu et al., 2009; Young, 1985a). Similarly, in
flatmounts of adult Crx>Math5 BAC retinas that were exposed to an EdU pulse at
E17.5, we observed very few, if any,EdU+ RGCs (Fig. V-5C). Therefore, the
envelope of RGCs births is not extended by prolonged Math5 expression.
Retroviral Math5 does not induce RGC fate or cell cycle exit in retinal
explants
We also tested whether Math5 can bias proliferating progenitors towards
RGC fate, using a retroviral vector to transduce cultured retinal explants. E13.5
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retinas were infected at low density ex vivo with MIG vectors. The resulting
single-copy proviruses express Math5 and GFP, or GFP alone from the potent
MSCV LTR promoter (Hawley, 1994) (Fig. V-6A). After 7 days in culture, we
counted the number of Brn3a+ RGCs (Fig. V-6B-C). Among 70 clones infected
with the IRES-GFP retrovirus, 4/272 GFP+ cells were Brn3a+ (1.5 0.7%
binomial SD, Fig. V-6D), in accord the fraction of RGCs produced by in vivo
clonal analysis (Turner et al., 1990). Among 60 clones infected with Math5IRES-GFP retrovirus, 6/262 GFP+ cells were Brn3a+, which is not different from
those transduced with GFP alone (2.2 0.9% binomial SD, Fishers exact
P = 0.34). Because overexpression of bHLH factors can promote cell cycle exit
and differentiation (Farah et al., 2000), we also evaluated clone size. We found
that the size distribution did not vary significantly between explants infected with
these viruses (Fig. V-6E, 2 test P = 0.6 for df = 4), indicating that high-level
single-copy expression of Math5 does not significantly promote cell cycle exit.
As a positive control, we also analyzed explants infected with an MSCV-IRESdnMAMLGFP retrovirus. The dnMAMLGFP fusion protein autonomously blocks
Notch signaling by interfering with the NICD-CSL transcriptional complex
(Maillard et al., 2004). Inhibition of Notch activity is associated with premature
cell cycle exit and stimulation of RGC fate among early progenitors (Austin et al.,
1995; Nelson et al., 2007). Among 52 clones, we detected a modest increase in
the fraction of Brn3a+ RGCs (Fig. V-6D, 3/60 GFP+ cells, 5 3%, P = 0.11). We
also observed a significant reduction in clone size, with all cells deriving from
one- or two-cell clones (Fig. V-6E, 2 test P < 10-8 for df = 4). These results
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confirm that blockade of Notch signaling by dnMAMLGFP drives cell cycle exit,
and that our explant system is sufficiently robust to detect this effect. Ectopic
Math5 expression in progenitors does not stimulate cell cycle exit or significantly
promote RGC fate in this system, consistent with our transgenic overexpression
findings (Fig. V-4).
Crx>Math5 expression partially rescues the RGC deficiency in Math5 KO
mice
Although Crx>Math5 Tg does not stimulate RGC genesis in the wild-type
environment, cells expressing ectopic Math5 may be prevented from adopting
the RGC fate by strong negative feedback from nascent RGCs (Austin et al.,
1995; Belliveau and Cepko, 1999; Waid and McLoon, 1998; Wang et al., 2005;
Zhang and Yang, 2001). Indeed, we have observed that many Brn3a+ are
generated in E13.5 Math5 KO retinal explants transfected with human ATOH7
(Prasov et al., 2012). We therefore crossed Crx>Math5 Tg and Crx>Math5 BAC
transgenes onto the Math5 KO background to examine the potential of Crxdriven Math5 to stimulate RGC genesis, heterochronically and heterotopically
(Fig. V-1A), in a deficient environment.
We evaluated retinal flatmounts for the density of mature RGC cell bodies,
axons and fascicles (Fig. V-7A-C). In Math5 heterozygous mice, the vast
majority of axons made radial projections to the optic disc and were fasciculated
(Fig. V-7A,B), and the GCL contained many Brn3a+ RGCs (Fig. V-7C). In
contrast, Math5 KO retinas had vastly reduced axon density, in accord with
previous estimates (Lin et al., 2004). The residual axons were largely
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unfasciculated and exhibited pathfinding defects similar to those in Brn3b -/- mice
(Badea et al., 2009; Gan et al., 1999) (Fig. V-7B and Fig. V-S10). Among 10
flatmounts examined from Math5 KO eyes, no Brn3a+ cells were observed in any
area (Fig. V-7C). The Crx>Math5 Tg transgene, and to a lesser extent the
Crx>Math5 BAC, were variably capable of rescuing axons and preventing
fasciculation defects in Math5 KO retinas. However, rescue was less
pronounced with successive generations of mice, ostensibly due to epigenetic
reductions in transgene expression levels (Garrick et al., 1998). The Crx>Math5
Tg, but not the Crx>Math5 BAC, transgene was capable of restoring Brn3a
expression among some adult ganglion cells. As expected, all rescued Brn3a+
ganglion cells were derived from progenitors that expressed the Crx>Math5 Tg
transgene (GFP+), whereas only ~40% of Brn3a+ cells were GFP+ in the wildtype (Fig. V-7D). Finally, we observed small optic nerves in rescued mice
carrying the Crx>Math5 Tg (Fig. V-7E), but not in Math5 KO controls. Together,
these results suggest that the Crx>Math5 Tg transgene, which expresses high
levels of Math5 in early post-mitotic precursors and low levels in progenitors, can
partially rescue RGC genesis and axonal guidance defects in Math5 KO mice.
Crx>Math5 expression alters the RGCs birth profile in Math5 KO mice
Given the differences in the timing of Crx and endogenous Math5
expression, we tested whether the rescued RGCs were born within the same
temporal envelope as native RGCs, which are only generated prenatally. We
first exposed P1 pups to a single pulse of BrdU, and stained their retinas at P21.
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differed significantly (2 test P = 0.01 for df = 2). In particular, very few RGCs
were generated in the rescued mice (Crx>Math5 Tg; Math5 KO) at the earliest
developmental times, and a larger number of RGCs were born at E15.5. Third,
RGCs were generated within the same temporal envelope in all 4 genotypes,
suggesting that this time window is fixed. It does not depend on Math5, and
cannot be shifted by protracted or elevated Math5 expression.
Fourth, the absolute number of rescued RGCs at P1 in Crx>Math5 Tg;
Math5 KO mice was increased 2.3-fold in comparison to control Math5 KO mice
(Welch t-test P = 0.02, Fig. V-8G), but this effect was variable, and the number of
RGCs was low in comparison to Math5 hetetozygotes (12%, 1160 250 Brn3a+
cells per mm2, P < 10-8). The Crx>Math5 transgene did not increase the RGC
density in Math5 hets (10,400 400 Brn3a+ cells per mm2 vs. 10,800 600,
Students t-test P = 0.54), consistent with the results above (Fig. V-4). Likewise,
the number of RGCs was significantly reduced in control Math5 KO mice
compared to heterozygotes (5%, 500 50 Brn3a+ cells per mm2, P < 10-8), as
previously reported (Lin et al., 2004).
Increased cell death and optic nerve defects in rescued Crx>Math5 Tg mice
The variability and incomplete rescue of RGCs in Crx>Math5 Tg mice on
the Math5 KO background was surprising. To explore the mechanism underlying
this variability, we analyzed optic nerve development during early
embryogenesis, using TuJ1 to identify RGC axons. At E15.5 and E17, we
observed coalescence of TuJ1+ fibers and formation of optic nerves in
Crx>Math5 Tg; Math5 KO, but not in Math5 KO retinas (Fig. V-9A,B). These
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rescued optic nerves were much thinner than wild-type controls. In some cases,
these nerves exhibited severe pathfinding defects as they exited the retina, and
formed large knot structures (Fig. V-9A, arrowhead). Because RGCs that fail to
properly establish connections in the CNS are eliminated (O'Leary et al., 1986),
we evaluated cell death by activated Caspase-3 immunostaining at E16.5, near
the end of RGC genesis. We observed a significant increase in cell death in
Math5 KO mice compared heterozygous controls (Fig 9C-D, 9 1 vs. 1.1 0.2
Casp3+ cells per field, Welch t-test P < 0.001), consistent with previous reports
(Feng et al., 2010). In Crx>Math5 Tg; Math5 KO mice, there was a further
increase in cell death compared to Math5 KO retinas (12.9 0.6 Casp3+ cells per
field, P = 0.016). The vast majority of dying cells were located in the GCL (Fig. V9C), suggesting that aberrant RGCs are eliminated. We also observed knots of
RGC axons in Crx>Math5 BAC; Math5 KO mice, consistent with the small degree
of rescue by this transgene (Fig. V-9E).
Discussion
The patterns of Crx>Cre BAC and Crx 2.4 kb transgene expression
The Crx 2.4 kb promoter has been used to drive Cre, lacZ, Nrl or Nr2e3
expression (Cheng et al., 2006; Furukawa et al., 2002; Koike et al., 2005; Nishida
et al., 2003; Oh et al., 2007), and Crx BACs have been used to drive GFP,
hPLAP or lacZ expression (Muranishi et al., 2010; Samson et al., 2009). From
these studies, it is clear that both types of transgenes are expressed at high
levels in photoreceptors and bipolar cells, and their precursors. However,
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the pineal, at least some transcription factors are likely to regulate both genes,
and bind within this 2.4 kb sequence. Indeed, the zebrafish Crx ortholog is
expressed in proliferating retinal progenitors (Shen and Raymond, 2004), and it
has been suggested that bovine Crx, along with Otx2, regulate gene expression
in the RPE (Esumi et al., 2009). Second, the level of expression was significantly
higher (16-fold) from the conventional transgene (Fig. V-2 and Fig. V-S3).
Together, the qualitative and quantitative differences we observed can explain
the greater abundance of GFP+ cells in Crx>Cre Tg mice compared to Crx>Cre
BAC mice.
We believe the patterns of cumulative transgene expression reflect
dichotomous low-level or leaky activity in proliferating RPCs, and high-level
activity in photoreceptor and bipolar precursors, for four reasons. First, Cre
immunoreactivity closely matched endogenous Crx expression, in adult and
embryonic retinas (Fig. V-2). Second, high-level Crx or Cre expression was
observed only in post-mitotic cells (Fig. V-S7 and data not shown) (Muranishi et
al., 2011). Third, GFP reporter expression was distributed in radial stripes in
BAC transgenic mice (Figs. 2-3 and Suppl Figs. 1,7), suggesting a clonal origin.
Fourth, low concordance (<40%) was observed between R26floxGFP and Z/AP
reporters in the GCL and inner INL of Crx>Math5 BAC and Crx>Cre BAC mice,
but high concordance (~100%) was observed in photoreceptor and bipolar
neurons. This dichotomy contrasts starkly with the uniformly high concordance
observed for a Math5>Cre BAC transgene (Brzezinski et al., 2012), and
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Crx>Math5 Tg mice at any point during development (Fig. V-4). It is likely that
negative feedback from nascent RGCs (Austin et al., 1995; Belliveau and Cepko,
1999; Waid and McLoon, 1998; Wang et al., 2005; Zhang and Yang, 2001)
restricts Math5 from inducing supernumerary RGCs. Second, the temporal
profile of RGC births was not extended by Crx>Math5 Tg expression (Figs. 5 and
8), despite abundant Crx expression in early post-mitotic precursors during the
post-natal period.
Third, retroviral expression of Math5 did not significantly promote RGC
fate or cell cycle exit in cultured embryonic retinal explants. These results differ
from previous studies in chick and frog, in which overexpression of Math5
orthologs favored RGC fate (Kanekar et al., 1997; Liu et al., 2001). In these
studies, effects on cell cycle dynamics and cell fate could not be completely
isolated. By itself, any experimental manipulation that forces cell cycle exit
during early neurogenesis can cause RPCs to adopt early fates, by effectively
stopping progression of the histogenetic clock (Ohnuma et al., 2002). Indeed,
high-level expression of proneural bHLH factors induces cell cycle exit in vitro
(Farah et al., 2000), and Xath5 overexpression reduces clone size in vivo (Moore
et al., 2002). Furthermore, misexpression of other bHLH factors, such as
Neurod1, during early frog development promotes RGC fate, whereas
overexpression of Xath5 during late developmental stages favors non-RGC fates
(Moore et al., 2002). In mice, Math5 is unlikely to be a major determinant of cell
cycle exit, because it is variably expressed, during or after the terminal division,
and lineage-marked cells do not re-enter the cell cycle in Math5 KO mice
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births in Math5 KO mice closely matches wild-type birthdating curves, except for
the extremely low RGC abundance (Fig. V-8). Together, these findings suggest
that the pattern of Math5 expression is not the sole factor restricting RGC
competence. Instead, a complex network of interactions, including Math5, is
likely to determine the spatiotemporal pattern of RGC genesis. For example, the
envelope of RGC genesis also appears to be shaped by other transcription
factors, Notch signaling, and microRNAs (Elliott et al., 2008; Georgi and Reh,
2010; Silva et al., 2003).
A pioneering model for RGC fate specification
In Crx>Math5 Tg; Math5 KO and Math5 KO mice, the loss of early-born
RGCs (Fig. V-8) is correlated with pathfinding defects in remaining RGCs (Fig. V9 and Fig. V-S10). These observations can be explained in two ways. First, the
residual and rescued RGCs may follow an aberrant RGC differentiation pathway.
In adult Math5 KO mice, residual RGCs form dendritic arbors with normal size
and spacing (Lin et al., 2004), but these cells fail to express Brn3b and Brn3a
(Fig. V-7 and data not shown), which are critical for axon pathfinding, dendritic
stratification, and cytodifferentiation (Badea et al., 2009; Gan et al., 1999). It is
thus possible that Crx>Math5 Tg derived RGCs are intrinsically defective and
express an aberrant set of RNA transcripts. Alternatively, the pathfinding defects
in these RGCs may result indirectly, from a deficiency of early-born ganglion
cells, which may limit the extent of rescue overall. Nascent RGCs are known to
elaborate signals, such as sonic hedgehog (Shh), which promote intraretinal
axon pathfinding generally (Erskine and Herrera, 2007; Oster et al., 2004). In
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zebrafish, the establishment of early RGC axons is necessary and sufficient for
pathfinding and survival of later RGCs (Pittman et al., 2008), and this community
effect may be widespread in the nervous system (Raper and Mason, 2010). In
Math5 KO mice, residual RGC axons are poorly fasciculated, and often branched
(Fig. V-7), and do not extend radially toward the central retina (Fig. V-S10). In
Crx>Math5 Tg; Math5 KO animals the extent of fasciculation is roughly correlated
with the number of surviving RGCs (data not shown). Thus, isotypic interactions
are likely to be critical for proper pathfinding and fasciculation of the transgenerescued RGCs. These pioneering effects are not limited to intraretinal
pathfinding, as knots of tangled fibers were apparent behind the retinas of
rescued Crx>Math5 Tg and BAC animals (Fig. V-9). These defects appear to be
resolved by apoptosis, although a small number of ganglion cells survive, and
are likely to make synaptic connections in the brain (Triplett et al., 2011). Our
results highlight the robust pathfinding mechanisms that operate in the retina,
and the strong homeostatic mechanisms that balance the ratio of diverse cell
types during development.
Acknowledgements
The authors are grateful to Thom Saunders, Maggie van Keuren and the UM
transgenic animal model core for generating conventional and BAC transgenic
animals; to Sue Tarl, Dellaney Rudolph, Christine Brzezinski and Melinda Nagy
for technical support; to Cheryl Craft for Crx antisera; to Sean Morrison and Ivan
Malliard for the MIG and dnMAML retroviral constructs, respectively; to Mitchell
Gillett for assistance with histology; to Anand Swaroop and Edwin Oh for Crx 2.4
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Figure V-1. Crx and Math5 (gal) are expressed in overlapping subsets of cells
shortly after cell cycle exit. (A) Schema comparing the termporal expression
patterns and cellular abundance of Crx (red) and Math5 (black) in the retina. (B-C)
Sections from E13.5 (B) or E15.5 (C) Math5-lacZ/+ embryos costained for gal
(Math5-lacZ allele), Crx, and EdU following a 4 hour chase in vivo. Many
cells coexpress Math5 and Crx (arrowheads), and both factors are expressed
in some cells shortly after cell cycle exit (EdU+, arrows). Cells initiating
expression of Crx or Math5 reflect similar populations of progenitors. RPE, retinal
pigmented epithelium; NBL, neuroblastic layer; GCL, ganglion cell layer. Scale bar,
50 m.
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Figure V-3. Widespread Crx>Math5 expression has little effect on cell fate
decisions in the retina. (A-F) Sections from adult Crx>Math5-IRES-Cre BAC
(shortened as Crx>Math5 BAC) or matched Crx>Cre BAC control transgenic
mice carrying R26floxGFP reporters were coimmunostained with GFP and
informative markers. GFP and marker costaining (bottom) and GFP alone (top)
are indicated (arrows). Mller glia were identified by Sox9 (A,B), amacrines by
AP2 (C,D) and horizontal neurons by calbindin (E,F). Arrowheads in E and F mark
calbindin+ horizontal cells that are GFP. (H-I) Flatmounts of adult Crx>Math5 BAC
and Crx>Cre BAC retinas with rhodamine dextran labeled RGCs. Some lineagemarked RGCs (arrows) and displaced amacrine cells (arrowheads) are indicated.
While both transgenes mark essentially all photoreceptors and bipolar cells (Fig.
V-S2), a small fraction of other cell types was also labeled in each case. The
fate spectra are similar, with or without Math5. Rare horizontal cells were
labeled in Crx>Math5 BAC Tg mice, but not in Crx>Cre BAC Tg controls, most
likely due to differences in transgene expression level. Scale bar, 50 m.
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Figure V-4. Widespread Crx>Math5 expression does not alter RGC abundance or
retinal histology. (A-C) Sections from E13.5 (A), P0 (B) and P22 (C) control or
Crx>Math5-IRES2-Cre transgenic (Crx>Math5 Tg) retinas stained for Brn3a (A,B) or
rhodamine dextran (C) to mark RGCs, and counterstained with DAPI to mark nuclei.
(D) RGC fraction among GCL neurons at P0 and P22. There is no significant
difference in the RGC fraction between transgenic (Tg) and contol retinas. (E) Low
(top) and high (bottom) magnification views of basic fuchsin- and methylene bluestained plastic sections. The retinal histology is similar in Crx>Math5 Tg and control
mice. Scale bar: 50 m in A-C; 100 m in E.
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Figure V-5. Widespread Crx>Math5 expression does not extend the profile of
RGC births, but decreases the numbers of early-born photoreceptors. (A)
Birthdating of Crx>Math5-IRES-Cre transgenic (Crx>Math5 Tg) and littermate
control retinas. Embryos were exposed to BrdU at E15.5 and analyzed at P22.
There were 2-fold fewer E15.5 birthdated cells in the ONL (photoreceptors) of
Crx>Math5 transgenic animals compared to controls, and corresponding
increases in the INL and GCL. (B) Crx>Math5 Tg pups were similarly exposed to
BrdU at P1 and harvested at P22. Few, if any, GCL cells were labeled with BrdU
in transgenic or control mice. (C) Crx>Math5-IRES-Cre BAC (BAC) embryos were
exposed to EdU at E17.5, and their RGCs were labeled with rhodamine dextran at
P22. No EdU+ RGCs were detected in central flatmounts of BAC or control
retinas. Prolonged transgenic expression of Math5 does not extend the RGC
birthdating profile. Scale bar: 100 m in A-B; 50 m in C.
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Figure V-6. Retroviral Math5 overexpression does not stimulate RGC fate
or cell cycle exit in retinal explant cultures. (A) Experimental design.
Retinas were explanted from E13.5 embryos, flattened on polycarbonate
membranes, infected at low density with the indicated MSCV retrovirus,
and cultured for 7 days in vitro (DIV). Isolated GFP+ clones were scored
for RGC number by Brn3a immunoreactivity and clone size. (B-C)
Example clones from explants infected with IRES-GFP (B) or Math5IRES-GFP (C) retroviruses. (D) Plot showing the fraction of GFP+ cells
that developed as Brn3a+ RGCs in transduced explants. There was no
significant difference in the RGC fraction of explants transduced with
Math5-IRES-GFP or IRES-GFP control. A modest increase in RGCs was
observed when Notch signaling was autonomously blocked in clones with
the IRES-dnMAML virus. Error bars show binomial standard deviation.
(E) Clone size distribution. There was no difference between Math5IRES-GFP and IRES-GFP explants, but clone size was significantly
reduced in explants infected with IRES-dnMAM. Scale bar, 50 m.
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Figure V-S2. Crx transgenic and BAC expression patterns in the pineal
gland, retinal pigmented epithelium, and ciliary body. (A) Pineal gland
sections from Crx>Math5 Tg and Crx>Math5 BAC mice stained for
R26floxGFP reporter expression. GFP is evident in the pineal gland, but
absent in surrounding connective tissue. (B) Sections of adult retinal
pigmented epithelium (RPE) from transgenic mice stained for the GFP
reporter. Although the signal is partially obscured by melanin, GFP is
abundant in conventional, but not in BAC transgenic mice. (C)
Immunofluorescence (top) and differential interference contrast (bottom)
images of P1 peripheral retina from a Crx>Math5 Tg; R26floxGFP pup.
GFP expression was apparent in both pigmented and unpigmented ciliary
body (CB) epithelial cells. NR, neural retina.
Scale bar, 50 m.
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Figure V-S3. Direct comparison of Crx>Math5 BAC and endogenous Crx transcript
levels. (A) Diagram of the triplex competitive RT-PCR strategy. Primers and expected
product sizes are shown within the Crx locus and Crx>Math5 BAC. A common forward
primer labeled with 6-FAM (Ex1) was used in the PCR. Reverse primers in Crx exon 2
(Ex2) or in Math5 (M5) were used in an equal molar ratio. (B) Capillary electrophoresis
profiles of triplex competitive PCR products amplified from Crx>Math5 BAC (top) or
wild-type (WT, bottom) adult retinal RNA templates. In Crx>Math5 BAC retinas (n = 5),
the molar ratio of transgenic Math5 and endogenous Crx transcripts, inferred from the
ratio of PCR products, was 533%, consistent with single-copy expression. This value
(k) was used to normalize qRT-PCR measurements (Fig. V-2D).
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Figure V-S4. Math5 does not grossly alter secondary fate choices of
photoreceptors and bipolar cells. (A-H) Sections from adult Crx>Math5-IRES-Cre
BAC (Crx>Math5 BAC) or matched Crx>Cre BAC control transgenic (Crx>Cre
BAC) mice carrying the R26floxGFP reporter were coimmunostained for GFP and
indicated photoreceptor or bipolar marker. In the ONL, cone subtypes were
identified by M- or S-opsin staining, and rods by the absence of staining (A-D). In
the INL, bipolar cells were identified by Chx10 staining (E-F), and rod bipolar
subtypes were identified by PKC staining (G-H). Virtually all photoreceptors and
bipolar cells are contained within the Crx lineage (GFP+). There is no apparent
difference in the distribution of these subtypes between transgenes. Scale bar, 50 m.
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Figure V-S9. Crx>Math5 Tg mice and control mice exhibit similar levels of
apoptosis throughout development. (A-D) Sections from E13.5, E15.5,
E17.5 or P0 Crx>Math5 transgenic (Tg) mice or control littermates were
stained with DAPI and an antibody to cleaved Caspase-3 to identify
apoptotic cells (arrows). Scale bar, 200 m.
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CAGCCCAGGCTTAAAGTCG (Crx ex1)
CGCCGCATGCAGGGGCTGAACACG
ATACCGGAGATCATGCAAGCTGGT
CGCCGCATGCAGGGGCTGAACACG
GCTCTTTTCCAGCCTTCCTT
CCTCCTGAATGACGCTAGGA
CCAAACTGGAACAACACTCAACCC
GATTGAGTTTTCTCCCCTAAGACCC
GTACTTGCGCTCAGGAGGAG
GAGTCTGGGACATGTTCAGTT
CCAAACTGGAACAACACTCAACCC
57
60
58
60
58
56
55
Annealing
temp$
1X Masteramp
N/A
1X Masteramp
1X Masteramp
Notes
45-60 sec
These reactions were done using a three-primer PCR, with a common 6-FAM labeled forward primer and equal ratios of reverse primers.
All PCRs had an initial denaturation step (94C x 3 min); followed by 40 cycles of 30-45 sec at 94C for denaturation,
at the noted temperature for annealing, and 1 min at 72C extension; with a final extension step (7 min at 72C)
Single-stranded oligos were used to delete Math5 and IRES sequence by recombineering, to generate the Crx>Cre Tg, and the Crx>Cre
BAC targetting construct
endogenous Math5
transgenic Math5
total Math5
actin
CTCTGTTCCTGCTTATTGGGG
RT-PCR
RT-PCR
RT-PCR
RT-PCR
ATACCGGAGATCATGCAAGCTGGT
Crx Tg genotyping
CCACAGTCTCTGAAGATCCTGTGATCTCGAAATTCACCATGGGCCCAAAGAAGAAGAGAAAGGTTTCGAA
TTCGAAACCTTTCTCTTCTTCTTTGGGCCCATGGTGAATTCGAAATAGGTCCCCTCACACGGGGCGACCT
Experiment
Table V-S1. Oligonucleotide primers and PCR conditions used in this study
CHAPTER VI
ATOH7 MUTATIONS CAUSE AUTOSOMAL RECESSIVE PERSISTENT
HYPERPLASIA OF THE PRIMARY VITREOUS
Abstract
240
explants. The R65G variant retains all of these activities, similar to wild-type
human ATOH7. Our results strongly suggest that arPHPV is caused by N46H
and is etiologically related to congenital retinal nonattachment (NCRNA). The
R65G allele, however, cannot explain the ONA phenotype. Our study firmly
establishes ATOH7 as a retinal disease gene and provides a functional basis to
analyze new coding mutations.
Introduction
The vertebrate neural retina is a highly ordered laminar structure, which
contains rod and cone photoreceptors, interneurons, specialized glia, and
projection neurons (Rodieck, 1998). During development, these diverse cell
types are generated in a fixed, but overlapping, temporal order from a pool of
multipotent neuroepithelial progenitors (Livesey and Cepko, 2001). At the onset
of retinal neurogenesis, on embryonic day E11 in mice and during the 5th
gestational week in humans, the first neurons exiting the cell cycle differentiate
as retinal ganglion cells (RGCs). Their axons form the optic nerves, which relay
all visual information to the brain.
The adult retina is nourished by two major vascular systems. The central
retinal artery enters the eye along with the optic nerve and supplies the inner
retina, while a tunic of choroidal vessels supplies the outer, photoreceptor layers
(Fruttiger, 2002; Gariano and Gardner, 2005). The developing retina and lens
primordia are perfused by a three-dimensional arterial plexus that branches from
the central hyaloid artery as it emerges from the optic stalk. The hyaloid arcades
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fill the vitreous and drain into annular vessels at the edge of the optic cup.
During midgestation in humans and early neonatal life in mice, these hyaloid
blood vessels and the associated pupillary membrane regress, and the central
artery remodels to form a two-dimensional branching network that originates at
the optic nerve head (Fruttiger, 2002; Gariano and Gardner, 2005). This
vascular lattice spreads radially across the retinal surface along a scaffold of
migrating astrocytes, which are activated by signals from nascent ganglion cells
(Fruttiger et al., 1996). A second, concentric wave of vessels then branches from
the surface plexus, penetrating to form two deep layers within the mature neural
retina.
The development of retinal neurons is likewise controlled in tandem by
intrinsic transcriptional programs and the microenvironment (Livesey and Cepko,
2001; Ohsawa and Kageyama, 2008; Yang, 2004). As an archetypal instrinsic
factor, the basic helix-loop-helix (bHLH) nuclear protein Atoh7 (Math5) is
transiently expressed in early retinal progenitors (Brown et al., 1998; Brzezinski
et al., 2012) and is necessary for RGC fate specification. Atoh7 -/- mice exhibit a
profound deficiency of RGCs and lack optic nerves (Brown et al., 2001; Wang et
al., 2001). In the absence of RGCs, the mature retinal vasculature fails to form.
Consequently, the hyaloid (fetal) vessels persist, proliferate, and invade the inner
retina (Brzezinski et al., 2003; Edwards et al., 2011). The structure, function, and
spatiotemporal expression of Atoh7 have been highly conserved during
metazoan evolution. Atoh7 was initially identified by its homology to the
invertebrate atonal (ato) and lin32 genes (Brown et al., 1998; Brzezinski et al.,
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Ethics statement
All human studies were approved by the University of Michigan
Institutional Review Board and conform to the Declaration of Helsinki (Khaliq et
al., 2001). Mouse studies were approved by the University of Michigan
Committee on the Use and Care of Animals.
Human subjects
PHPV pedigree.
245
including clinical phenotypes of five blind individuals (Khaliq et al., 2001). None
of these patients had documented light perception, and all exhibited gross
nystagmus. In the youngest patient (VI-2), retinal folds were noted in one eye,
but no optic discs were seen during an exam under anesthesia, due to the
presence of a dense white-gray fibrous mass in both vitreae, which obscured the
central fundus. Unfortunately, the optic nerve status of affected individuals could
not be assessed, and they were not available for orbital MRI examination or other
clinical follow-up.
ONA cases.
perception. Fundus and MRI exams revealed complete absence of optic nerves,
chiasm, and optic tracts. She developed normally until 8 months, but after 12
months, was at the 5th percentile for height and weight. She exhibited severe
developmental delays in spoken language and other milestones, and at 8 years
of age had persistent difficulties initiating social interactions, an inability to
sustain conversations, poor motor planning and processing, and deficits in
sensory integration and auditory processing. Both parents have normal vision
and cognition. Relevant clinical features of four unrelated ONA patients are
listed in Table VI-S1, including cases 2-4, which were previously reported
(Brodsky et al., 2004; Lee et al., 1996; Scott et al., 1997). Each patient has
bilateral aplasia of the optic nerves, chiasm and tracts, with variable
retinovascular fndings. There was no consanguinity or family history of ocular
disease.
246
ONH cases.
247
Plasmid vectors
Full-length human ATOH7 and E47 cDNAs were subcloned in pCS2 or
pCS2-MT vectors (Turner and Weintraub, 1994) with the simian cytomegalovirus
IE94 enhancer/promoter driving expression. For electroporation experiments,
the ATOH7 coding sequence was subcloned in the pUS2-MT-IRES-GFP vector
(Zhang et al., 2012) with an N-terminal 6X-myc epitope tag (MT) and the human
ubiquitin C promoter (UbC) driving expression of a bicistronic transcript that
encodes MT-ATOH7 and GFP.
248
Expression plasmids carrying ATOH7 variants were constructed by sitedirected mutagenesis using the overhanging primer method (Liu and Naismith,
2008) with reagents and conditions noted in Table VI-S2. Reactions were
performed in 1X Pfu Ultra reaction buffer (Stratagene, Santa Clara, CA) with 0.4
M primers, 0.2 mM dNTPs, and 2.5 U of Pfu Ultra HF polymerase.
Masteramp (Epicentre, Madison, WI) was added at 20% (v/v) to melt
secondary structure in the GC-rich ATOH7 cDNA template (Prasov et al., 2010).
Products were digested with DpnI and transformed into E. coli DH5. The
resulting clones were verified by restriction analysis and DNA sequencing.
Electrophoretic mobility shift assays (EMSA)
Nuclear extracts were prepared and electrophoretic mobility shift assays
were performed using established methods (Hellman and Fried, 2007; Wadman
et al., 1997). Briefly, HEK293T cells were transfected in 6 cm plates using
FuGene6 reagent (Roche, Indianapolis, IN) with 1 g plasmid DNA, consisting of
an equal-ratio mixture (1:1) of wild-type or variant pCS2-ATOH7 and pCS2-E47
expression plasmids, or pCS2 empty vector. After 48 hrs, nuclear extracts were
prepared following lysis in cold 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5
mM DTT, 0.5% (w/v) NP40, pH 8.0, and centrifugation at 4000 g for 0.5 min.
The nuclear pellets were resuspended in cold 20 mM HEPES, 1.5 mM MgCl2,
420 mM NaCl, 0.2 mM EDTA, 25% (v/v) glycerol, pH 8.0 and agitated vigorously
for 30 min. The protein content of soluble nuclear lysates was estimated by
Bradford assay (Bradford, 1976). Ten micrograms of each extract was mixed
with 3 U poly[dI-dC] (Sigma, St. Louis, MO) in binding buffer (20 mM HEPES, 50
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250
251
252
253
Results
ATOH7 mutation segregates with PHPV disease
A locus for autosomal recessive persistent hyperplastic primary vitreous
(arPHPV) was mapped in a large consanguineous pedigree to a 13 cM region in
10q21 (Khaliq et al., 2001). This segment contains ATOH7 and the critical region
for nonsyndromic congenital retinal nonattachment (NCRNA), a clinically related
recessive disorder (Ghiasvand et al., 2000) (Fig. VI-1A). In a recent study, we
showed that NCRNA is most likely caused by a 6.5 kb deletion, located 21 kb
upstream from the ATOH7 start site, which removes a transcriptional enhancer
with three evolutionarily conserved noncoding elements. The NCRNA patients
are blind from birth, with no light perception, and have bilateral profusions of
retrolental fibrovascular tissue, similar to arPHPV patients (Khaliq et al., 2001).
They also have optic nerve aplasia, documented by magnetic resonance imaging
(MRI), and early bilateral detachments of the retina, with an apparent tractional
basis. In both diseases, the anterior chambers are shallow and there is
progressive corneal opacification, most likely due to chronic endothelial blood
staining (Brodrick, 1972).
Given the similarity between disease phenotypes, overlap of the linkage
intervals including ATOH7, and potential for shared ancestry between ethnic
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255
p.Arg65>Lys (R65G) amino acid change (Fig. VI-2). This was confirmed by
restriction analysis of PCR products, and was paternally inherited. A similar
R65G variant was previously reported in a heterozygous Australian child among
12 cases of isolated optic nerve hypoplasia (Macgregor et al., 2010).
Given the potential association between ATOH7 and optic nerve
hypoplasia, we also screened 30 patients with severe isolated ONH for ATOH7
coding mutations. No additional variants were found. Taken together, the
frequency of the ATOH7 R65G among optic nerve aplasia or hypoplasia patients
is estimated to be 2 out of 94 chromosomes. This variant was identified in 7 of
6592 chromosomes from individuals with normal vision, including 0/144 in
control chromosomes, 6/4924 in the exome variant database (Project), and
1/1524 reported by Macgregor et al. (Macgregor et al., 2010). Thus, R65G is
statistically overrepresented among ONA and ONH patients (Fishers exact test,
P = 0.007).
Copy number variant (CNV) analysis
In addition to sequencing of ATOH7 coding and regulatory regions, we
performed high-density SNP analysis to evaluate CNVs among the ONA
patients. In Patient 1, this revealed an 828 kb duplication that is predicted to
disrupt the CNTN4 (contactin) gene (Suppl. Fig 1). We mapped this duplication
by junctional PCR and determined that it was a precise tandem duplication
encompassing exons 2-12 (Fig. VI-S2A-C). Furthermore, in a lymphoblastoid
cell line from Patient 1, this allele can generate a truncated CNTN4 mRNA
transcript by premature polyadenylation, with termination after exon 12 (Fig. VI-
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258
We next modeled the ATOH7 bHLH domain (Fig. VI-3B) using the known
crystal structure of the NeuroD1-E47 heterodimer bound to DNA (Longo et al.,
2008). In this homology model, the Asn46 side group makes direct contact with
a thymine base in the core E-box recognition site (position 5 of CANNTG).
Introduction of a histidine residue at this position is predicted to significantly
impair DNA binding (Fig. VI-3C, bottom). In contrast, the neighboring Ala47 side
chain does not directly contact DNA, but the threonine substitution may alter
DNA binding properties through conformational effects. The positively charged
Arg65 residue, located at the end of Helix 1, is predicted to interact with the
negatively charged Asp61 side group via an electrostatic bridge, which may
serve to stabilize the helix or limit the flexibility of tertiary interactions (Kumar and
Nussinov, 2002) (Fig. VI-3C, top).
Given the proximity of N46 and A47 residues, and direct contact between
N46 and DNA (Fig. VI-2C), we tested the ability of these variants to bind an Ebox DNA recognition site (Fig. VI-4A). Mammalian plasmid expression vectors
for wild-type or variant ATOH7 proteins were transfected into HEK293T cells, and
the resulting nuclear extracts were compared in an electrophoretic mobility shift
assay (Fig. VI-4B). We also tested a humanized version of the zebrafish lakritz
mutation (p.L56>P), which is believed to cause a complete loss-of-function (Kay
et al., 2001). Because neurogenic bHLH transcription factors bind DNA as
heterodimers with ubiquitous class A bHLH proteins (Murre et al., 1989), we
included an expression plasmid for the binding partner E47 in some
transfections, to augment the low endogenous level of E47 in the HEK293T cell
259
line. Heterodimeric complexes between specific class A bHLH proteins and E47
assemble on DNA and do not form in solution (Wendt et al., 1998). Although this
binding of heterodimers is strongly favored, the E47 protein can also bind DNA
as a homodimer, with a characteristic gel shift pattern corresponding to different
phosphorylated isoforms (Sloan et al., 1996).
The radiolabeled synthetic double-stranded oligonucleotide probe
contained an E-box binding site (CAGGTG) that is optimal for ATOH7 and its
orthologs (Del Bene et al., 2007; Powell et al., 2004) (Fig. VI-4A). In the absence
of added E47, each ATOH7 variant, including wild-type, failed to bind DNA (Fig.
VI-4B). In the presence of wild-type ATOH7, faster migrating ATOH7-E47
complexes are the dominant species bound to DNA. The R65G and A47T
variants formed ATOH7-E47 heterodimers on DNA that were indistinguishable
from wild-type (R65G) or slightly reduced (A47T). These alleles therefore retain
DNA-binding and dimerization activity in vitro. In contrast, extracts containing
N46H or L56P variants formed only E47 homodimeric complexes, indicating that
these mutants are unable to bind DNA. Because the E47 homodimeric
complexes were not disrupted, N46H and L56P do not act as dominant-negative
proteins, like the Id class of bHLH factors (Benezra et al., 1990).
Western analysis confirmed that equivalent levels of ATOH7 and E47
were present in nuclear extracts, suggesting that the ATOH7 variant proteins
have similar stability (Fig. VI-4C). To explore this point further, we performed a
cycloheximide pulse-chase experiment (Zhou, 2004) (Fig. VI-S4). Because each
260
protein variant decayed at approximately the same rate, the observed DNAbinding impairment cannot be attributed to decreased protein stability.
We next tested the transcriptional activity of ATOH7 variants in a
luciferase cotransfection assay using HEK293T cells and an optimized synthetic
reporter, which contains a multimerized E-box sequence (CAGGTG) and minimal
-actin promoter (Akazawa et al., 1995; Flora et al., 2007) (Fig 4D). Wild-type
ATOH7 increased luciferase activity 25-35 fold compared to empty pCS2 vector,
in a dose-dependent manner (Fig. VI-4E). The A47T variant had an intermediate
level of activity compared to wild-type, 60% at the high dose (100 ng) and 20% at
the low dose (20 ng). R65G was indistinguishable from wild-type at the high
dose, but had 60% activity at the low dose. In contrast, N46H and L56P variants
had no detectable activity. Taken together, these results suggest that ATOH7
N46H and L56P are null alleles, whereas A47T and R65G retain significant
function.
261
retinas for 3 days in vitro (DIV) to allow for RGC differentiation. To assess the
degree of rescue, we immunostained whole explants for GFP, the RGC nuclear
marker Brn3a (Nadal-Nicolas et al., 2009; Xiang et al., 1995), and the
pan-neuronal marker 3-tubulin (TuJ1) (Fig. VI-5B). We then counted the
fraction of transfected (GFP+) cells that developed as ganglion cells (Brn3a+)
(Fig. VI-5C). In explants electroporated with the control vector (GFP only), very
few Brn3a+ cells were detected among the GFP+ cohort (7 3%) and very few
GFP+ TuJ1+ axons were apparent, as expected (Fig. VI-5, Fig. VI-S5A). In
contrast, the majority of cells transfected with the wild-type ATOH7 vector
adopted the RGC fate (72 12% Brn3a+), demonstrating a wide dynamic range
for this assay. Moreover, large bundles of GFP+ TuJ1+ processes, likely
representing RGC axon fascicles, were readily observed in these explants at low
magnification (Fig. VI-S5B). Explants transfected with N46H or L56P variants
failed to develop RGCs (P < 10-5 compared to wild-type, Fig. VI-5C) and could not
be distinguished from the negative control (GFP only), by RGC number (Fig. VI5B,C) or axon density (Suppl. Fig 5C,D). In contrast, R65G and A47T variants
did restore RGC development (Fig. VI-5B,C, Suppl. Fig 5E,F). These results
support our conclusion that N46H and L56P variants are functional null alleles,
while R65G and A47T retain complete or partial biological activity.
Discussion
ATOH7 has an established role RGC development (Brown et al., 2001;
Wang et al., 2001) and a secondary role in retinal vascular development
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cognate E-box recognition site (Fig. VI-4), and has no biological function in
restoring RGC development (Fig. VI-5).
Sixth, Atoh7 -/- mice have eye phenotypes that are similar to human
PHPV. In mice, Atoh7 is exclusively expressed by cells in the neural retina
(Brzezinski et al., 2012) and the primary pathology in Atoh7 mutant mice is loss
of RGCs (Brown et al., 2001). However, major vascular defects occur as a
secondary consequence of the ganglion cell deficiency, because RGCs are vital
for proper migration and development of retinal astrocytes (Fruttiger et al., 1996),
which form a scaffold for the growth of intrinsic retinal blood vessels (Fruttiger,
2002). In Atoh7 -/- mice, the intrinsic vasculature fails to develop, and hyaloid
(fetal) vessels persist in the vitreous and proliferate to supply the retina
(Brzezinski et al., 2003; Edwards et al., 2011). These abnormal vessels are
prone to hemorrhage, in the subretinal and intravitreal space, and some
extravasated blood communicates with the anterior chamber (hyphema).
Similarly, in the arPHPV family, all affected family members were blind from birth
and had bilateral retrolental masses (Khaliq et al., 2001). Persistent hyaloid
vessels were clearly observed in the youngest patient. In addition, the adult
patients had anterior chamber pathology, including cataracts, corneal opacity and
anterior synechiae, which may be related to chronic intraocular hemorrhage
(Brodrick, 1972).
Seventh, allelic ATOH7 variants have been identified in three families with
similar retinovascular pathology (Ghiasvand et al., 2011; Khan et al., 2011). In
NCRNA disease, linkage, genomic, and transgenic analysis suggest that a 6.5 kb
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266
conserved and important for DNA binding (Atchley and Fitch, 1997; Chien et al.,
1996; Jarman et al., 1993). Our results, however, suggest that an Ala47 Thr
substitution can be tolerated. In contrast, Asn46 makes direct contact with a
DNA base, and is critical for binding and function. In Drosophila, the orthologous
residue is one of three that are altered in the ato1 mutation (Jarman et al., 1994).
Our findings suggest that the Asn Ile substitution at this position is the primary
defect in the ato1 allele.
The ATOH7 R65G variant is predicted to destabilize Helix 1 based on
molecular modeling (Fig. VI-3) and is classified as a potentially damaging variant
based on bioinformatic criteria (Macgregor et al., 2010). However, the R65G
variant had a surprisingly small effect on protein function and stability in vitro, and
on biological function ex vivo. Likewise, the L56P substitution in the zebrafish
lakritz variant behaved unexpectedly. Introduction of a proline at this position is
predicted to disrupt Helix 1 and decrease the overall stability of the protein.
Instead, the humanized lakritz polypeptide was stable (Fig. VI-S1), but failed to
complex with the E-box recognition site (Fig. VI-4), due to impaired dimerization
with E47 or DNA binding.
In principle, variants that fail to bind DNA, such as L56P and N46H, could
act as dominant-negative proteins, similar to the Id class of bHLH factors
(Benezra et al., 1990). Id proteins lack DNA-binding activity, but sequester and
inhibit the function of other bHLH factors (Norton, 2000; Ross et al., 2003). In
contrast, our EMSA results, and the unaffected status of lakritz and arPHPV
heterozygotes, suggest that L56P and N46H mutations are simple null alleles.
267
Acknowledgements
The authors are grateful to the patients and families for participating in the
study; to Nathan Vale, Dellaney Rudolph and Susan Tarl for technical support;
to Ingrid Scott, Roberto Warman and Bronwyn Bateman for providing blood
samples from optic nerve aplasia patients; to Bob Lyons, Susan Dagenais and
the University of Michigan DNA core for assistance with the Illumina Omni1Quad
SNP analysis; to Chris Edwards and the UM microscopy and image analysis
laboratory staff for technical advice; to David Turner and Huanqing Zhang for
advice on retinal explants electroporation and for providing the pUS2-MT-IRESGFP vector; to Adriano Flora and Huda Zoghbi for the (E-box)x7 luciferase
reporter plasmid; to Tomomi Kaneko-Goto and Yoshihiro Yoshihara for sharing
Cntn4/BIG-2 mutant mouse tissues; to the Autism Genetic Resource Exchange
(AGRE) consortium for comparative genotyping data; to Donna Martin, Anthony
Antonellis, Chris Chou and David Turner for valuable discussions and critical
reading of the manuscript. The research was funded by grants from The
268
WEB RESOURCES
NHLBI Exome Variant Database
http://evs.gs.washington.edu/EVS/
www.ncbi.nlm.nih.gov/dbvar/
http://www.ncbi.nlm.nih.gov/genome/
assembly/2928/
SWISS-MODEL server
http://swissmodel.expasy.org/
269
Figure VI-1. The ATOH7 p.Asn46>His allele segregates with autosomal recessive
persistent hyperplastic primary vitreous (arPHPV) disease. (A) Minimal region of
shared homozygosity for arPHPV on chromosome 10q21, between D10S1221 and
GATA121A08 (Khaliq et al., 2001). The ATOH7 gene (red) and NCRNA critical
region (Ghiasvand et al., 2000) (yellow) are indicated. The same distal flanking
marker (GATA121A08 or D10S1418) delimits both intervals. (B) ATOH7 sequence
chromatograms from arPHPV family members. The AAC to CAC mutation causes an
Asn46His amino acid substitution (N46H) in the bHLH domain and loss of an EaeI
restriction site (underlined). This variant is heterozygous in the obligate carrier (IV 5)
and homozygous in the affected individual (V 5). (C) Map of the ATOH7 cDNA,
showing the informative PCR product and EaeI restriction fragments. (D) Agarose
gel of EaeI-digested PCR products amplified from arPHPV family members,
numbered as previously reported (Khaliq et al., 2001). Filled and half-filled symbols
show affected indivduals, and carriers assigned by haplotype (Khaliq et al., 2001),
respectively. Carriers have both mutant (270 bp) and wildtype (198 and 72 bp)
alleles, whereas blind individuals have only the mutant allele. NCRNA,
nonsyndromic congenital retinal nonattachment; bHLH, basic helix-loop-helix domain;
wt, wildtype; mut, mutant.
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Figure VI-2. The ATOH7 p.Arg65>Gly allele in a child with optic nerve aplasia
and developmental delay. (A) Sequence chromatogram showing the
heterozygous AGG to GGG variant in this case (Patient 1). The variant causes
an Arg65Gly (R65G) amino acid substitution, and creates a BstUI restriction site
(underlined) in the PCR product. (B) Map and acrylamide gel autoradiograph
showing wild-type and mutant BstUI-digested PCR products. The forward PCR
primer was end-labeled with 32P, giving wildtype (wt, 208 bp) and mutant (mut,
48 bp) radioactive fragments, respectively. The R65G allele was paternally
inherited.
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272
273
Figure VI-4. The arPHPV mutant ATOH7 polypeptide (N46H) does not bind
DNA or activate transcription, while A47T and R65G variants retain these
functions. (A-C) Electrophoretic mobility shift assay (EMSA). (A) Sequence of
EMSA probe radiolabeled with 32P--dCTP (red). The core bHLH recognition site
(E-box) is highlighted. (B) EMSA autoradiogram comparing DNA-binding activity.
HEK293T cells were cotransfected with plasmids encoding wildtype (WT) or
variant ATOH7 proteins, or empty vector (pCS2), with or without heterodimeric
bHLH partner E47. Nuclear extracts were incubated with dsDNA probe and
electrophoresed through a 6% acrylamide gel. In the absence of ATOH7, native
and phosphorylated E47 isoforms (67 kDa) bind DNA as a homodimer, giving a
pattern with two major complexes (E47-E47). In the presence of wildtype
ATOH7 (17 kDa), faster-migrating E47-ATOH7 complexes predominate. ATOH7
variants N46H (arPHPV) and L56P (corresponding to lakritz) fail to bind DNA,
such that only E47 homodimers are observed in these lanes. In contrast, R65G
and A47T variants form ATOH7-E47 heterodimers. Addition of ATOH7 blocking
antibody (Ab) or cold competitor oligo (cc) decreases the amount of probe bound
by the wildtype ATOH7-E47 complex. (C) Western blots of EMSA nuclear
extracts probed with ATOH7 or E47 antibodies show similar levels of these
proteins. (D-E) Luciferase cotransfection assays. (D) The reporter contains 7
tandem E-box sites and a minimal -actin promoter driving expression of firefly
luciferase. (D) Comparison of ATOH7 transcriptional activity. HEK293T cells
were cotransfected with Renilla control and firefly luciferase reporters and
varying doses of ATOH7 expression vectors. Firefly luminescence counts,
normalized to Renilla, are reported as fold activity relative to pCS2 vector. The
N46H and L56P variants have no detectable transcriptional activity, and were
significantly different from wild-type (t-test P < 0.03 for 100 ng and P < 3 x 10-6 for
20 ng). The A47T and R65G variants are not significantly different from wild-type
at the high plasmid dose (100 ng, 60% and P = 0.09 for A47T, 100% and P = 0.77
for R65G), but have reduced activity at the low dose (20 ng, 20% and P < 10-5 for
A47T, 60% and P < 0.001 for R65G). Error bars show the standard deviation of
three replicates from a representative experiment. prom, promoter.
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Figure VI-5. Human ATOH7 R65G and A47T variants rescue ganglion cell
specification in Atoh7 -/- retinal explants, but N46H and L56P mutants do not.
(A) Experimental design. Eye cups from E13.5 Atoh7 -/- embryos were
electroporated ex vivo with a DNA solution containing bicistronic expression
plasmids pUS2-myc-ATOH7-IRES-GFP (encoding wild-type or variant ATOH7
proteins) or pUS2-myc-IRES-GFP (negative control). After a 6-hour recovery
period, retinas were explanted onto polycarbonate membranes, cultured for 3
days in vitro (DIV), fixed and immunostained as wholemount preparations. (B)
Confocal images of ATOH7-transfected retinal explants stained with GFP and
Brn3a antibodies to mark RGCs. In the absence of Atoh7 function, few RGCs
are formed (GFP only). Wild-type (WT), R65G and A47T variants rescue RGC
development in the transfected cell cohort, while N46H and L56P variants do not.
(C) Quantitative analysis of the RGC fraction among transfected cells (GFP+).
Data from two experiments (open and closed circles) are plotted together, with
each point representing a different explant. The mean and SD are indicated.
Each variant was compared to GFP-only or wild-type ATOH7 controls by
Students t-test, with P-values listed below the graph. N46H and L56P mutants
induce significantly fewer RGCs than wild-type ATOH7, and are indistinguishable
from the GFP-only control. IRES, internal ribosome entry site; myc, 6X myc
epitope tag; RGC, retinal ganglion cell. Scale bar, 50 m.
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Figure VI-S1. ONA Patient 1 carries a duplication that disrupts CNTN4. (A)
LogR ratio and B allele frequency plots show Illumina Omni1-Quad SNP
genotyping for the patient and her unaffected mother. The 0.83 Mb duplication
on chromosome 3p26 is shaded in red. The B-allele frequency splitting and logR
ratio increase are characteristic of a 3-to-2 copy gain, which was not maternally
inherited. (B) CNTN4 (contactin-4) genomic region. The duplication in patient 1
(thick red line) spans exons 2-12 and is not present in the database of normal
genomic structural variants. However, other duplications (red) and deletions
(green) overlap CNTN4 in patients with autism spectrum disorder, as reported in
the indicated publications. Human genome coordinates are based on NCBI build
36 (hg18). Mb, megabase.
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Figure VI-S4. ATOH7 variants have similar protein stability. (A) LICOR dual
fluorescence Western blots of HEK293T cells transfected with wildtype or
variant ATOH7 expression plasmids. Lysates were harvested after 0-5 hours
treatment with cycloheximide (CHX), which blocks synthesis of new proteins.
The level of ATOH7 polypeptide was normalized to endogenous_tubulin, which
has a long half-life. (B) Quantitative analysis of ATOH7 levels in (A) based on
LICOR antibody fluorescence (semilog plot). The variant (red) and wildtype
(black) ATOH7 proteins have equivalent decay kinetics.
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283
284
Sex
Patient
Number
no
yes
no
bilateral micropthalmia,
iris coloboma (OD),
pigmented epithelium
mottling, choroidal
neovascularization
unilateral
microopthalmia and
microcornea(OS);
chorioretinal atrophy;
bilateral iris colobomas
yes
bilateral
microphthalmia;
retinochoroid
depigmentation, absence
of retinal vasculature,
abnormal vessels at area
of optic disc
bilateral
microphthalmia,
bilateral iris coloboma
no
Septo-optic
dysplasia
none
Other ocular
abnormalities
none
none
none
posterior pituitary
ectopia; absence of
pituitary infundibulum;
hypothyroid, pituitary
insufficiency
failure to thrive
Endocrine or pituitary
defects
none
none
none
1.2 MB hemizygous
deletion at 14q23
(includes OTX2)
developmental delay;
delayed vocalization;
auditory processing
defects
hypoplastic corpus
callosum
Genetic findings
Neurological or brain
defects
this study
this study
Reference
285
nested
nested
AGACCACTCCCTGCCAATGT
AGACAGCGTTGTTTGGCATC
TGTGTTCCTTTCTTAGTTTGATATGGT
TTTGCTATAGTTTGTCATTTTTGCTT
GGCCACGCGTCGACTAGTAC
GGCCACGCGTCGACTAGTAC
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT
3RACE RT
N/A
ACCAACACTGAACTCTTCACCT
CCAGTGTCACAGGAATGTGG
CCACTGGGGAACCACCCCGCGTAAGCGGTCGAAG
GCGGCGGCGCTCGCGCGCGTGGGCCGCCAGGCGCCTG
ATGCGGCGGCGCTCGCGCGTGTTGGCCGCCAGGCGCCT
GCGGTCGAAGGCAGTGTTGGGCCCCTGCATGCGGCGG
CNTN4 WT
TCCAGGTGGTGGGTAAAAGA
CTTCGACCGCTTACGCGGGGTGGTTCCCCAGTGG
CNTN4 duplication
CCTGGCGGCCCACGCGCGCGAGCGCCGCCGCATGCAG
GCCTGGCGGCCAACACGCGCGAGCGCCGCCGCATGC
CGCATGCAGGGGCCCAACACTGCCTTCGACCGCTTAC
Experiment
Table VI-S2. Oligonucleotide primers and PCR conditions used in this study
50C for 1 hr
Notes
Cycling conditions
CHAPTER VII
DISCUSSION AND FUTURE DIRECTIONS
The results in this thesis provide novel and important insights into the
structure and function of Math5 (Atoh7), the mechanism of RGC fate of
specification, and the role of ATOH7 in human disease. My work has opened up
many new exciting avenues for future directions. My work highlights the
importance of careful experimentation, proper interpretation of findings, and
consideration of alternative hypotheses. In testing new ideas and hypotheses, I
have often uncovered flaws in previous studies, such as the discovery of splicing
of Math5 mRNA (Kanadia and Cepko, 2010) discussed in Chapter II. In each
case, I identify logical explanations for the discrepancies between our findings
and the interpretations of other research groups. Lasting conclusions in science
depend on reproducible results, which come from multiple lines of converging
evidence. As such, there are many future directions that could expand and
strengthen the conclusions presented in this dissertation.
The structure of Math5 (Atoh7)
In Chapters II and VI, we examined the gene, mRNA, and protein
structure of Math5. One observation in these studies was the high G+C content
and secondary structure in the 3 terminus of the Math5 RNA transcript, which
was the likely explanation for the PCR and Northern blotting artifacts observed by
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Kanadia and Cepko (2010). While this secondary structure is unlikely to disrupt
transcription or RNA processing, Math5 mRNA may be difficult to translate, and
this may provide an additional level of regulation. Indeed, we observed that fulllength Math5 protein could not be produced in E. coli (data not shown). In
eukaryotes, secondary structure and high G+C content may impair translation
(Baim et al., 1985; Kenneson et al., 2001). Further work is necessary to establish
whether translation of Math5 mRNA is a regulated step in retinal progenitors, and
if this step is hypersensitive to ribosome functional status. In principle, impaired
translation of Math5 mRNA could contribute to the severe optic nerve hypoplasia
observed in the setting of Rpl24 riboprotein mutation in Bst/+ mice (Oliver et al.,
2004). This could be tested directly using a Math5-HA knock-in allele (Fu et al.,
2009) in Bst heterozygous mice, or with transfection assays in Bst/+ or wild-type
fibroblasts in vitro.
The fate plasticity of Math5 (Atoh7) cells
In this thesis, I have explored the plasticity of the Math5-expressing cells
(Chapter III and V) and the timing of onset of Math5 and RGC factor expression
relative to the cell cycle (Chapters III and IV). These studies suggest that the
Math5 population is heterogeneous in many aspects, including fate choice and
onset of expression. Indeed, we observed that the Math5>Cre expressing
population can generate all 7 major cell types on the same day during
development. However, it remains unclear whether single Math5+ cells are
multipotent at a given time during development or are heavily biased (restricted)
in their fate selection. Furthermore, it remains to be determined whether the
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288
expect only two clone sizes from MADM analysis using Math5>Cre. Precursors
in which Cre is expressed in post-mitotic (G0) cells would result in a single-cell
clones that are yellow (G1 recombination pattern). In contrast, progenitors in
which Cre is expressed during late S or G2 of the last cell division would
generate two-cell clones with one red and one green cell (X segregation), or
single-cell clones with one yellow and one unmarked cell (Z segregation). If
larger clone sizes are observed, this would necessarily mean that Math5expressing cells continued to cycle. Clone size could also be assessed in Math5
-/- mice, in order to determine whether lineage cells re-enter the cell cycle.
In principle, the fates of daughters in informative two-cell clones could be
concordant or discordant, and may include RGCs or other cell types. Based on
our clonal analysis of explants, many progenitors that give rise to RGCs generate
pairs of this cell type (Chapter IV). These data are seemingly discordant with
previous clonal analysis of frog and rodent retinas (Holt et al., 1988; Turner et al.,
1990), as no definitive two-RGC clones were detected in these studies. Four
major differences between our study and these prior analyses could account for
this discrepancy, and these could largely be reconciled by the in vivo MADM
analysis. First, analyses were carried out in mature adult retinas, after the period
of post-natal RGC culling (Farah and Easter, 2005). Thus, some RGCs among
paired clones likely died during development. Second, due to technical reasons,
progenitors could only be infected at E13.5 or later in the mouse, a time at which
many RGCs have already exited the cell cycle. Thus, RGCs were
underrepresented in clones compared to their contribution to the retina (Jeon et
289
al., 1998; Turner et al., 1990). Third, due to tangential dispersion (Reese and
Galli-Resta, 2002) or cell migration, some RGC clones could not be definitively
assigned. Fourth, species specific differences may result in different clonal
properties between frogs and rodents (Holt et al., 1988).
MADM analysis with Math5>Cre would circumvent each of the limitations.
As Math5>Cre is active at the onset of neurogenesis, in vivo clonal analysis
would follow the earliest cells as they exit mitosis. Coupled with EdU birthdating,
clones could be stratified based on time of cell cycle exit to precisely determine
the fate spectrum Math5+ cells during development. Because clones are sparse
and daughters in two cell clones are marked with different colors, clones could be
unambiguously assigned even in the presence of cell migration. Furthermore,
RGC fate could be scored in flatmounts at P0, prior to the period of post-natal
culling. In the adult, the localization of reporter expression coupled with the
characteristic morphologies and laminar positions of each of the major cell type
would allow for unambiguous scoring of 2-cell clones. MADM analysis could also
be done in Math5 -/- mice, in order to thoroughly evaluate the secondary fate
choices of cells in the Math5 lineage. Taken together, this single experiment
could confirm or challenge our previous findings, and would provide definitive
and novel insights into the fate choices of Math5-expressing cells, the cell cycle
timing of Math5 expression, and the frequency of symmetric RGCs clones.
The non-autonomous role of Math5-expressing cells
Though RGC development requires the function of Math5 (Brown et al.,
2001; Wang et al., 2001), we found that only 55% of RGCs are contained in the
290
Math5 lineage (Chapter III). Furthermore, an even smaller percentage (only 2030%) of the earliest born cohort of RGCs expresses Math5, yet nearly all of these
cells are lost in Math5 -/- mice. Together, these results suggest a nonautonomous role of Math5 in the specification of RGCs. It is likely that a
secreted or cell surface factor is made by Math5 cells, but not by neighboring
neurogenic cells in the early retina.
Two sets of experiments could be used to confirm this role of Math5 and
also to identify the precise factors involved. First, analysis of chimeric mice
carrying a mix of Math5 -/- and reporter-marked wild-type cells could provide
confirmation that Math5 cells have a non-autonomous role in RGC development.
Specifically, in chimeras composed predominantly of wild-type cells, we could
determine whether Math5 -/- cells are more likely to adopt the RGC fate. In
reciprocal chimeras, we would expect the opposite results, i.e. wild-type cells in a
Math5 -/- environment, cannot form mature RGCs. Alternatively, Math5 could be
conditionally deleted or overexpressed using a mosaic Cre, such as Chx10>Cre
(Rowan and Cepko, 2004). Second, microarray analysis could be conducted to
determine the gene expression profiles of flow-sorted early Math5-expressing
progenitors, using Math5>GFP (Hufnagel et al., 2007) or ATOH7-3034>mCherry
(Ghiasvand et al., 2011) transgenic mice. These profiles could be compared in
Math5 -/- and +/+ backgrounds, to determine what specific factors are lost in
Math5 -/-. This analysis would provide candidate genes and factors that nonautonomously regulate RGC development in the early E11.5-E12.5 retina.
291
292
(Liu et al., 2000). However, definitive Brn3b lineage and over-expression studies
in the mouse are lacking. To prove that Brn3b is a marker of committed RGCs,
the descendents of Brn3b cells could be traced using a Brn3b>Cre BAC
transgenic mouse, similar to the tracing experiments presented in Chapter III. It
is expected that lineage-marked cells only contain ganglion cells. To establish
the role of Brn3b in biasing RGC fate, Brn3b could be expressed in the Math5
pattern, using a Math5>Brn3b-IRES-Cre BAC transgene. In this way, lineal
descendents of Brn3b over-expressing cells could be followed by crossing to
floxed reporters. It is expected that Brn3b would bias Math5 lineage cells
towards RGCs and suppress amacrine fates. Together, these studies would
determine if Brn3b is sufficient for stimulating ganglion cell production in
competent progenitors and if it is a master regulator of RGC development.
Testing the pioneering model of RGC fate
In Chapter V, I thoroughly characterized the birthdates of ganglion cells in
Crx>Math5 Tg; Math5 -/- mice. I found that high-level expression of Math5 did
not extend the profile of RGC births, and that early born ganglion cells were
largely lost in these mice. This event was correlated with pathfinding defects,
RGC death, and incomplete rescue. These results support a pioneering model of
RGC fate in the mouse, as is evident in zebrafish (Pittman et al., 2008).
However, causation could not be definitely established due to confounding
variables in the experiment. Thus, a cleaner experiment could directly test the
pioneering model of RGC development in mice, and determine the critical
embryonic times for this pioneering. In principle, the critical window of pioneering
293
RGC axons may be related to the closure of the optic fissure, which occurs
during early development (Barishak, 1992; Gregory-Evans et al., 2004). This
critical time period could be determined using an inducible Math5 expression
system in the context of a Math5 -/- genotype, i.e. with a dual transgenic Tet-On
system (reviewed in (Corbel and Rossi, 2002)). While this may be cumbersome,
Math5 expression, and thus the window of RGC births, could be precisely
controlled and delayed. It is expected that induction at E11.5 would result in
complete rescue, while induction at later developmental time points (e.g. E14.5)
would rescue initial specification, but not terminal differentiation of RGCs.
The role of ATOH7 (Math5) in human disease
Data presented in Chapter VI, together with recent studies (Ghiasvand et
al., 2011; Khan et al., 2011), have established a role for ATOH7 in congenital
neurovascular diseases of the retina. Causative mutations in ATOH7 were
identified in a group of related developmental disorders, including non-syndromic
congenital retinal non-attachment (NCRNA, Ghiasvand et al., 2011), vitreoretinal
dysplasia (VRD, Khan et al., 2011), and persistent hyperplastic primary vitreous
(PHPV, Chapter VI). These diseases all share common pathogenetic features
and an overlapping clinical spectrum of symptoms. The common genetic feature
among patients is a homozygous loss-of-function mutation in ATOH7. Thus, the
underlying cause of disease is likely the agenesis of the optic nerve. This was
not appreciated in many of these patients, because retinovascular disease
prevented visualization of the fundus (Ghiasvand et al., 1998; Khaliq et al.,
2001). Given the wide spectrum of symptoms in among NCRNA, VRD, and
294
295
296
Figure VII-1. Outline of the Mosaic Analysis of Double Markers (MADM) strategy.
(A) The MADM technique consists of two reciprocally chimeric reporters.
Recombination can occur in G2, and be resolved by X or Z segregation. In X
segregation, a single cell is double-colored. In Z segregation, both daughter cells are
labeled, with one functional reporter active in each (one green and one red cells).
Recombination can also occur in G1 or post-mitotic cells, which generates doublecolored cells. (B) Example of sparse-labeling in pancreatic islet cells using MADM
strategy with Ngn3>Cre driver. Images adapted from Zong et al., 2005 and Desgraz
and Herrera, 2009.
297
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